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• W2154411206 abstract "Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores in which the Ca2+ storage protein, chromogranin A (CGA), couples with InsP3-gated Ca2+ channels (InsP3R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP3R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP3Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP3R channel activity by CGA appears to be an essential component in the control of intracellular Ca2+concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis. Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores in which the Ca2+ storage protein, chromogranin A (CGA), couples with InsP3-gated Ca2+ channels (InsP3R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP3R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP3Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP3R channel activity by CGA appears to be an essential component in the control of intracellular Ca2+concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis. chromogranin A inositol 1,4,5-trisphosphate InsP3R receptor phenylmethylsulfonyl fluoride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid CGA1 is a member of the granin protein family and is stored in high concentrations in the large dense core secretory granules of most endocrine and neuroendocrine cells as well as in many nerve cells in the periphery and brain (1Simon J.P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, 2Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (611) Google Scholar). CGA, the first member of the granin family to be discovered (3Helle K.B. Mol. Pharmacol. 1966; 2: 298-310PubMed Google Scholar, 4Blaschko H. Comline R.S. Schneider F.H. Silver M. Smith A.D. Nature. 1967; 215: 58-59Crossref PubMed Scopus (409) Google Scholar, 5Smith A.D. Winkler H. Biochem. J. 1967; 103: 483-492Crossref PubMed Scopus (262) Google Scholar), has a wide variety of functions, both extracellular and intracellular. As one of its extracellular functions, CGA acts as a prohormone, a protein that contains numerous sites for proteolytic processing. Following secretion, extracellular proteases cleave CGA, generating several peptide fragments with biological activity, including pancreastatin (6Tatemoto K. Efendic S. Mutt V. Makk G. Feistner G.J. Barchas J.D. Nature. 1986; 324: 476-478Crossref PubMed Scopus (619) Google Scholar, 7Lewis J.J. Zdon M.J. Adrian T.E. Modlin I.M. Surgery. 1988; 104: 1031-1036PubMed Google Scholar), vasostatins I and II (8Drees B.M. Rouse J. Johnson J. Hamilton J.W. Endocrinology. 1991; 129: 3381-3387Crossref PubMed Scopus (98) Google Scholar, 9Aardal S. Helle K.B. Elsayed S. Reed R.K. Serck-Hanssen G. J. Neuroendocrinol. 1993; 5: 405-412Crossref PubMed Scopus (173) Google Scholar, 10Helle K.B. Serck-Hanssen G. Aardal S. Neurochem. Int. 1993; 22: 353-360Crossref PubMed Scopus (18) Google Scholar), parastatin (11Fasciotto B.H. Trauss C.A. Greeley G.H. Cohn D.V. Endocrinology. 1993; 133: 461-466Crossref PubMed Scopus (0) Google Scholar), catestatin (12Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Wu L. Taupenot H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (336) Google Scholar), and chromacin (13Strub J.M. Sorokine O. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1997; 272: 11928-11936Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In healthy individuals, CGA and its peptide fragments are present in the circulatory system in low nanomolar quantities. However, in patients suffering from pheochromocytoma and other neuroendocrine tumors, concentrations are significantly higher (14Aardal S. Aardal N.P. Larsen T.H. Angeletti R.H. Stridsberg M. Taupenot L. Aunis D. Helle K.B. Scand. J. Clin Lab. Invest. 1996; 56: 511-523Crossref PubMed Scopus (14) Google Scholar). Elevated plasma levels of CGA are associated with a number of pathological conditions making the protein an ideal marker not only for neuroendocrine tumors but also for chronic heart failure and brain disorders such as Parkinson's and Alzheimer's diseases (15Helle K.B. Aunis D. Adv. Exp. Med. Biol. 2000; 482: 389-397Crossref PubMed Google Scholar). Among its intracellular roles, CGA has been shown to interact with ATP, catecholamines, and Ca2+ (16Kopell W.N. Westhead E.W. J. Biol. Chem. 1982; 257: 5707-5710Abstract Full Text PDF PubMed Google Scholar, 17Daniels A.J. Williams R.J. Wright P.E. Neuroscience. 1978; 3: 573-585Crossref PubMed Scopus (69) Google Scholar), to acidify the intravesicular medium and to sort proteins for the regulated secretory pathway via a range of protein-protein interactions (15Helle K.B. Aunis D. Adv. Exp. Med. Biol. 2000; 482: 389-397Crossref PubMed Google Scholar). These sorting functions include aggregation with chromogranin B, complexing with dopamine β-hydroxylase, t-plasminogen activator, and binding secretory granule membrane constituents such as the InsP3R (15Helle K.B. Aunis D. Adv. Exp. Med. Biol. 2000; 482: 389-397Crossref PubMed Google Scholar). In recent years, secretory granules of neuroendocrine cells have been identified as inositol (1,4,5)-trisphosphate (InsP3)-sensitive Ca2+ stores (18Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar, 19Gerasimenko O.V. Gerasimenko J.V. Belan P.V. Petersen O.H. Cell. 1996; 84: 473-480Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 20Nguyen T. Chin W.C. Verdugo P. Nature. 1998; 395: 908-912Crossref PubMed Scopus (160) Google Scholar). In the granules CGA forms a tetramer and appears to bind four molecules of the intraluminal loop of the InsP3R at the intravesicular pH 5.5 (21Yoo S.H. Lewis M.S. J. Biol. Chem. 1992; 267: 11236-11241Abstract Full Text PDF PubMed Google Scholar, 22Thiele C. Huttner W.B. J. Biol. Chem. 1998; 273: 1223-1231Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23Yoo S.H. Trends Neurosci. 2000; 23: 424-428Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In vitro studies show that purified InsP3R interact directly with CGA at this pH and dissociate from it at pH 7.5, a pH encountered when exocytosis occurs (24So S.H. Yoo S.H. Kweon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Co-transfection of InsP3R and CGA into COS-7 cells followed by co-immunoprecipitation demonstrates that these two proteins form a complex in vivo (24So S.H. Yoo S.H. Kweon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We have investigated the functional aspect of this coupling via InsP3-mediated Ca2+ release studies using InsP3R-reconstituted liposomes in the presence and absence of CGA. We have further characterized the molecular basis of this phenomenon at the single channel level using planar lipid bilayer studies. In the presence of CGA the open probability and mean open time of the InsP3R channel increases significantly. Hence, modulation of InsP3R channel activity by CGA appears to be an essential component in the control of intracellular Ca2+concentration in secretory granules. The type I InsP3 receptor was isolated from bovine cerebella as described previously (25Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, bovine cerebella were mixed with 3 volumes of buffer I (50 mm Tris-HCl, pH 7.4, 0.32 m sucrose, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, 10 μmpepstatin), homogenized, and centrifuged at 2000 × gfor 10 min at 4 °C. The supernatants were re-centrifuged at 105,000 × g for 1 h to precipitate the membrane pellet, which was resuspended in buffer II (50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, 10 μm pepstatin) containing 1% Triton X-100, stirred for 1 h, and then centrifuged at 32,000 × g for 1 h at 4 °C. The resulting supernatant was mixed with an equal volume of buffer III (20 mm Tris-HCl, pH 7.5, 0.1m NaCl, 1% Triton X-100, 1 mmβ-mercaptoethanol, 0.1 mm PMSF, 10 μmleupeptin, 10 μm pepstatin), and applied to an InsP3R antibody-coupled immunoaffinity column (0.35 × 1 cm) equilibrated with 20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm CaCl2. The protein-loaded column was washed with 20 bed volumes of this buffer, and the InsP3R was eluted by 10 ml of elution buffer (0.1m glycine, pH 2.8, 0.2% Triton X-100, 0.5 mNaCl, 1 mm β-mercaptoethanol, 0.1 mm PMSF, 10 μm leupeptin, 10 μm pepstatin). The eluate was immediately neutralized by adding 1 m Tris-HCl, pH 9.5, and mixed with an equal volume of buffer IV (50 mmTris-HCl, pH 8.0, 0.2% Triton X-100, 0.5 m NaCl, 1 mm β-mercaptoethanol); it was then applied to a benzamidine-Sepharose column equilibrated with Buffer V (20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mKCl, and 3 m urea). InsP3R containing flow-through was collected and stored at −70 °C. until use. The type I InsP3receptor was solubilized in 1% CHAPS and purified from mouse cerebellum using heparin affinity and concanavalin A-Sepharose column chromatography as described previously (26Thrower E.C. Mobasheri H. Dargan S. Marius P. Lea E.J. Dawson A.P. J. Biol. Chem. 2000; 275: 36049-36055Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The purified InsP3R was then incorporated into liposomes by adding 15 μg of purified protein to 1 ml of liposome solution (consisting of phosphatidylcholine in bilayer buffer), mixing, and then incubating on ice for 10 min. InsP3R proteoliposomes were formed as described previously (25Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Some of these proteoliposomes had CGA encapsulated in them, and the remainder was used for control experiments. Ca2+ efflux from the proteoliposomes was measured by observing changes in indo-1 fluorescence. Fluorometric measurements were carried out at 35 °C using a Shimadzu RF-5301 PC spectrofluorometer equipped with a temperature-controlled cuvette holder. Fluorescence intensity was measured at the emission wavelength of 393 nm (excitation of 355 nm) with 10 nm of excitation band slit width and 10 nm of emission band slit width. For the kinetic analysis of InsP3-induced Ca2+ release, the data were acquired every 20 ms after each addition of the indicated InsP3 concentration to 0.5 ml of the proteoliposome solution. The fluorescent intensities of indo-1 were calibrated to free Ca2+ concentrations using a Ca2+-EGTA buffering system (27Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (396) Google Scholar). InsP3 dose-dependent Ca2+ release was also measured by the intensity of indo-1 fluorescence after each InsP3 addition and was compared with the fluorescence intensity after the addition of Triton X-100 instead of InsP3. In these experiments, 10 μm indo-1 was used, which is a high enough concentration to buffer released Ca2+, thus precluding the possibility of Ca2+regulation of the InsP3-induced release. Planar lipid bilayers were formed by painting a solution of phosphatidylethanolamine/phosphatidylserine (3:1; 30 mg/ml in decane) across a 100 μm aperture in a Teflon sheet bisecting a Lucite chamber. The hole was pre-painted with phosphatidylcholine/phosphatidylserine (3:1) prior to membrane formation. The two compartments are defined as cis(corresponding to the cytosol) and trans (corresponding to the lumen of the ER). The cis (cystolic) compartment consisted of 250 mm HEPES, Tris, pH 7.35, 0.5 mm EGTA ([Ca2+]free = 200 nm), ATP 0.5 mm, and ruthenium red 2 μm. The trans (luminal) compartment consisted of 250 mm HEPES, adjusted to pH 5.5 (as purified InsP3R was used in these experiments, the pH could be changed using 70 mm HCl), and 53 mmBa(OH)2. Single channel currents were amplified using a bilayer clamp amplifier (Warner Instruments) and recorded on digital tape. Data was filtered with an eight-pole Bessel filter to 500 Hz, digitized to 2 kHz, transferred to a personal computer, and analyzed using the pClamp 6.0 (Axon Instruments) software package. InsP3R proteoliposomes were added to the ciscompartment and mixed followed by the addition of 2 μmInsP3 to the same compartment. Upon InsP3R activation, single channel activity was recorded. CGA (1 μg) was added to the trans compartment and mixed. InsP3R single channel activity was recorded. The pH inside thetrans compartment was changed by adding Tris (final concentration 110 mm) to pH 7.5 (to dissociate CGA from InsP3R), and InsP3R single channel activity was recorded. These experiments were repeated (i) in the presence of increasing doses of InsP3 (over the range of 0.2–2 μm) to thecis compartment and (ii) at a fixed InsP3concentration of 2 μm in the presence of increasing free Ca2+ concentrations (over the range of 0.01–1 μm) to the cis compartment. Both steps i and ii were carried out in the presence and absence of 1 μg of CGA in thetrans compartment, and InsP3R single channel activity was recorded. The effect of CGA on InsP3 dose response for type I InsP3R from bovine cerebella was investigated initially using Ca2+ release studies. InsP3-induced Ca2+ release from InsP3R-reconstituted liposomes was monitored both in the presence and absence of CGA (Fig. 1). InsP3-induced Ca2+ efflux through the proteoliposomes (300 μm Ca2+ inside) was determined by the change of indo-1 fluorescence at 393 nm. The total amount of Ca2+ in the liposomes was determined by adding 1% Triton X-100, and this was the value set at 100%. Given this information, the total amount of InsP3-releasable Ca2+ was estimated to be 60%. When CGA was present inside the vesicle at pH 5.5, the pH value at which CGA associates with the InsP3R, InsP3-induced Ca2+ release was significantly enhanced (see Fig. 1a). AKapp value for InsP3 of 0.2 μm was obtained. When the pH was maintained at 7.5, however, the fluorescent changes seen at each InsP3 dose more closely resembled those seen in the absence of CGA at pH 5.5 (Kapp values for InsP3 of 0.8 and 0.9 μm, respectively), further supporting the pH dependence of the InsP3R/CGA interaction. The presence of CGA at pH 5.5 markedly increased the apparent affinity of the receptor for InsP3 when compared with the Ca2+ release obtained in the absence of CGA. This result complements the effect of CGA on InsP3 binding to its receptor (25Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Even at InsP3 concentrations lower than those published previously (starting at 0.05 μm) (25Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), an increase in apparent affinity for InsP3 was seen (Fig. 1b). The enhancement of Ca2+ release from InsP3R-reconstituted liposomes as a result of the pH-dependent interaction of CGA with InsP3R, illustrates a functional phenomenon associated with this coupling. To further define the actual mechanism of action, we investigated these effects at the single channel level using InsP3 R incorporated into planar lipid bilayers. Under control conditions, in the absence of luminal CGA and in the presence of cytosolic free Ca2+ (300 nm) and InsP3 (2 μm), mouse InsP3R type I, single channel activity was observed (see Fig.2a, trace i). Single channel currents of ∼2 pA were seen, and the presence of a pH gradient between the trans and cis compartments (pH 5.5:pH 7.35) did not affect channel activity. Two populations of mean open times were seen with values of 0.864 ± 0.039 and 8.84 ± 0.014 ms (Fig.3a). The data set is further expanded (Fig. 3b) to emphasize the complete population of longer open times. The open probability (Po) was 4.0 ± 1.0% (S.E.) (n = 4).Figure 3Mean open times for InsP3R in the presence and absence of CGA. a, mean open times for InsP3R in the absence of CGA. Two populations of open times are observed with values of 0.864 ± 0.039 and 8.84 ± 0.014 ms. b, an expanded section with a fit to the data. This experiment is typical of four similar but separate experiments.c, mean open times for InsP3R in the presence of CGA. Again, two populations of open times are observed with values of 2.61 ± 0.024 and 103.5 ± 0.003 ms, but now the number of longer openings has increased, and the mean open time is greater.d, an expanded section with a fit to the data. The increase in the number of longer open times is illustrated clearly. This experiment is typical of four similar but separate experiments.e, mean open times for InsP3R following dissociation of CGA by pH change (pH 5.5 to pH 7.5). Two populations of open times are observed with values of 1.05 ± 0.014 and 7.19 ± 0.013 ms, and the open times have returned to control levels.f, an expanded section with a fit to the data (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Less than 1 min after the addition of 1 μg of CGA to thetrans compartment, a dramatic change in channel activity was observed (Fig. 2a, trace ii). The magnitude of the single channel current remained unaltered; however, significant differences were apparent in mean open times andPo. Two populations of mean open time were evident, as before, but were greatly increased over control values (Fig. 3c). Values of 2.61 ± 0.024 and 103.5 ± 0.003 ms were obtained, illustrating an approximate 3-fold and 12-fold increase of open time in each respective population. This is displayed even more clearly when the scale from Fig. 3c is expanded to focus on the population of longer open times (Fig. 3d). Furthermore, a large increase in Po over control levels was seen with a value of 33.0 ± 8.5% (S.E.) (n = 4). By changing the pH of the trans compartment to 7.5, a condition known to cause dissociation of CGA from InsP3R, channel activity essentially reverted to control levels (Fig.2a, trace iii). The two populations of mean open time were reduced to 1.05 ± 0.014 and 7.19 ± 0.013 ms (n = 4, Fig. 3e, and compare with Fig.3a), and the Po was reduced to 3.0 ± 1.6% (S.E.), a value close to that seen for the control. The addition of heparin, an InsP3R-specific antagonist, to the cis compartment inhibited channel activity completely. A similar study was carried out using microsomes from mouse cerebellum to see whether the effects of CGA would still be seen in native tissue the same as in purified protein. These experiments were complicated by the fact that HCl was present in the transcompartment, a condition necessary for maintaining the pH at 5.5. Thus chloride channels present in the microsomes were activated, making the analysis difficult. Nonetheless, under comparable control conditions to those described for the purified receptor, the open probability was 8%; and upon addition of CGA to the transcompartment this increased to 52%. Altering the pH of thetrans compartment lowered the open probability to ∼1%. The addition of CGA to the trans compartment in the absence of InsP3R had no effect upon the bilayer itself, and CGA did not potentiate any InsP3R channel activity in the absence of cytosolic InsP3. Furthermore, the addition of 1 μg of CGA to the cis compartment in the presence of InsP3R and InsP3 did not affect channel activity. As CGA is a highly charged protein, control experiments were carried out to exclude the possibility that any charged macromolecule could be responsible for the effects observed in this study. Heparin is one such charged macromolecule and is known for its inhibitory effects on the InsP3R when exposed to the cytosolic face of the receptor, although it is not known to bind to its luminal face. The addition of 1 μg of heparin to the trans compartment (see Fig.2b) did not alter channel open probability (5% for the control compared with 4.4% in the presence of luminal heparin), indicating that CGA does have a specific modulatory effect on InsP3Rs. Single channel activity as a function of InsP3 concentration, both in the presence and absence of CGA, was characterized next (Fig.4, a and b). Over a range of InsP3 concentrations starting at 0.2 μm, the open probability was greater in the presence of CGA, with a 14-fold increase observed at 2 μmInsP3. When the pH of the trans compartment was changed to 7.5, the Po was reduced to that seen in the control experiments. The results obtained from the single channel experiments concur with those seen in the Ca2+release studies (see Fig. 1) in that, at each InsP3concentration and in the presence of CGA, a significant increase inPo is concomitant to an increased amount of released Ca2+. The Ca2+-dependence of the InsP3R was investigated in the absence and presence of CGA (Fig. 5, a and b) at a fixed InsP3 concentration of 2 μmand over a Ca2+ concentration range of 0.01–1.0 μm. As the Ca2+ concentration in thecis compartment increased successively from 0.01 to 0.3 μm, the Po increased, reaching a maximum value of 4% at 1 μm Ca2+(pCa 6) in the absence of CGA (Fig. 5b). At Ca2+ concentrations higher than 0.3 μm(pCa 6.5), no inhibition was seen (see Fig. 5b,expanded section). This lack of inhibition by free Ca2+ has been observed previously for purified receptor (28Thrower E. Lea E. Dawson A. Biochem. J. 1998; 330: 559-564Crossref PubMed Scopus (23) Google Scholar, 29Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and contrasts to the inhibition seen with microsomes (30Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1435) Google Scholar). Repetition of this experiment, with the addition of CGA to thetrans compartment, produced dramatic increases in channel activity (Fig. 5a). At a Ca2+ concentration of 0.01 μm (pCa 8) the Pois effectively zero in the absence of CGA, in contrast to thePo observed when CGA is present, which expressed as a percentage of total open probability is 34%. Furthermore, thePo effectively remains at this level irrespective despite increasing Ca2+ concentrations, and again no inhibition by Ca2+ is seen. The activating phase of the Ca2+ dependence seen in the absence of CGA is not apparent in its presence. The channel has reached maximal open probability at pCa 8. Thus, the lack of dependence on Ca2+ for activation in the presence of CGA and 2 μm InsP3 is clearly illustrated. Secretory granules of endocrine and neuroendocrine cells have been shown to serve as InsP3-sensitive intracellular Ca2+ stores (18Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 13446-13448Abstract Full Text PDF PubMed Google Scholar, 31Blondel O. Bell G.I. Seino S. Trends Neurosci. 1995; 18: 157-161Abstract Full Text PDF PubMed Scopus (47) Google Scholar), and additional evidence from goblet cells has demonstrated their direct participation in the control of cytoplasmic Ca2+ (20Nguyen T. Chin W.C. Verdugo P. Nature. 1998; 395: 908-912Crossref PubMed Scopus (160) Google Scholar). Chromogranins are Ca2+storage proteins that are found in secretory granules in millimolar concentrations (1–2 mm) (1Simon J.P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, 32Winkler H. Westhead E. Neuroscience. 1980; 5: 1803-1823Crossref PubMed Scopus (371) Google Scholar), and CGA binds 32 mol of Ca2+/mol with a dissociation constant of 2.7 mmat pH 7.5 and 55 mol of Ca2+/mol with a dissociation constant of 4 mm at pH 5.5 (33Yoo S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar). Given the high capacity Ca2+ binding of CGA, most of the 40 mmintravesicular Ca2+ remains bound, thus yielding a total free Ca2+ concentration of ∼24 μm inside the granules (34Bulenda D. Gratzl M. Biochemistry. 1985; 24: 7760-7765Crossref PubMed Scopus (74) Google Scholar). As secretory granules occupy about 10% of the total cell volume (at least in bovine chromaffin cells) (32Winkler H. Westhead E. Neuroscience. 1980; 5: 1803-1823Crossref PubMed Scopus (371) Google Scholar) and have a high storage capacity for Ca2+, they may play an important role in governing intracellular Ca2+ dynamics. This hypothesis is further supported by the discovery that chromogranins A and B interact directly with type I InsP3Rs located on the secretory granule membrane (24So S.H. Yoo S.H. Kweon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and that this coupling is functional, at least in terms of CGA (25Yoo S.H. Jeon C.J. J. Biol. Chem. 2000; 275: 15067-15073Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The present results obtained from our bilayer studies reemphasize the functional importance of the interaction and provide the first mechanistic insight at the level of a single InsP3R. The effects of CGA on InsP3R channel activity, at the intravesicular pH of 5.5 (the pH at which the coupling occurs), are very profound. The increase in both mean open time and open probability (Fig. 3) demonstrates clearly that CGA causes the channel to open more frequently and, once open, to stay open for longer times, which when translated to the cellular level, implies a greater release of Ca2+. When the intravesicular pH is altered to 7.5, the effects of CGA are seen to dissipate almost instantly (Fig. 2, trace iii, and Fig. 3,e and f) and resemble more closely those seen at pH 5.5 in the absence of CGA. The InsP3 concentration dependence both in bilayer studies and flux studies carried out at comparable concentrations complement one another. At the single channel level the InsP3R is seen to have a greater chance of opening when CGA is present, and in the flux studies, the apparent affinity for InsP3 is greater. Again, a change in pH to 7.5 causes the effect to revert to control levels. As for Ca2+dependence, the InsP3R is already active at maximal levels when CGA and InsP3 are present even when the level of cytosolic free Ca2+ is only 10 nm (Fig. 5). The data shown in Figs. 4 and 5 indicate that in the presence of coupled CGA and a sufficient concentration of InsP3 (2 μm), the characteristic effects of cytosolic Ca2+ on the InsP3R channel disappear. It appears that the conformation of the InsP3R is in such a state in the CGA-coupled condition, that high InsP3dictates the channel activity regardless of the presence of Ca2+. How do these results relate to the physiological situation? In the presence of CGA at intravesicular pH 5.5, the InsP3R is primed to respond to low levels of InsP3. Hence, when a secretory granule reaches the surface of the cell and docks with the inner surface of the plasma membrane, it is fully loaded with Ca2+ and sensitized for release. Generation of InsP3, even in small quantities, will cause a large elevation in local Ca2+ concentration (values as high as 100 μm have been seen immediately prior to exocytosis) (23Yoo S.H. Trends Neurosci. 2000; 23: 424-428Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), initiating secretory processes. During exocytosis, the vesicle contents are exposed to the extracellular pH of 7.4, thus causing dissociation of CGA from the InsP3R resulting in altered channel properties and hence Ca2+ release. Following these events, vesicular contents dissociate from the vesicle membrane, with secretory cargo moving to the extracellular space and then into the bloodstream. The chromogranins, particularly CGA, appear to play a role in intracellular Ca2+ dynamics and secretion, which also has significant implications to human disease. For example, patients suffering from pheochromocytoma and other neuroendocrine tumors have significantly higher concentrations of CGA measured in the plasma (14Aardal S. Aardal N.P. Larsen T.H. Angeletti R.H. Stridsberg M. Taupenot L. Aunis D. Helle K.B. Scand. J. Clin Lab. Invest. 1996; 56: 511-523Crossref PubMed Scopus (14) Google Scholar,15Helle K.B. Aunis D. Adv. Exp. Med. Biol. 2000; 482: 389-397Crossref PubMed Google Scholar). This high plasma CGA indicates that high levels must have been stored in order to be released. Individual cells contain increased CGA, and there are more CGA-containing cells. In various cholestatic liver diseases, a striking increase is seen in the number of bile ductules (35Roskams T. De van den Oord J.J. Vos R. Desmet V.J. Am. J. Pathol. 1990; 137: 1019-1025PubMed Google Scholar). These reactive bile ductules differ from their normal counterparts in that they display neuroendocrine features, in particular the expression of CGA. Hence, the presence of elevated CGA can lead to abnormal Ca2+ release within these cells, thus activating specific metabolic mechanisms in the cell, which leads to unnecessary growth. The increased release of neuroendocrine substances, including CGA, may also play an autocrine or paracrine regulatory role in ductular metaplasia of hepatocytes or bile ductule growth. Thus, although the role of CGA in disease is largely attributed to extracellular CGA and its derivatives, a contribution may arise also from intragranular CGA with respect to its effects on intracellular Ca2+ release and exocytosis. In conclusion, our present work is the first electrophysiological study detailing the very important physiological phenomenon arising from the interaction between CGA, a Ca2+ storage protein, and an intracellular Ca2+ release channel, InsP3R. We have shown that there is an order of magnitude increase in the open probability and open time of the InsP3-gated Ca2+ channel. These effects of the interaction of CGA and InsP3R have implications for exocytosis and neuroendocrine cell function in disease." @default.
• W2154411206 created "2016-06-24" @default.
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