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• W2072484997 abstract "Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+] i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+] i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+] i, they showed contrasting sensitivities depending on whether the [Ca2+] i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+] i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+] i at the plasma membrane as distinct from global changes in [Ca2+] i within the cytosol. Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+] i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+] i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+] i, they showed contrasting sensitivities depending on whether the [Ca2+] i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+] i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+] i at the plasma membrane as distinct from global changes in [Ca2+] i within the cytosol. The jellyfish protein aequorin with its co-factor, coelenterazine, has been used for over 3 decades to measure cytosolic calcium ([Ca2+] i) 1The abbreviations used are: [Ca2+] i, cytosolic calcium; HEK 293 cells, human embryonic kidney cells; ACVI, adenylyl cyclase type VI; ACVI/AEQ, adenylyl cyclase/aequorin chimera; cytAEQ, HA1-tagged aequorin; PCR, polymerase chain reaction. 1The abbreviations used are: [Ca2+] i, cytosolic calcium; HEK 293 cells, human embryonic kidney cells; ACVI, adenylyl cyclase type VI; ACVI/AEQ, adenylyl cyclase/aequorin chimera; cytAEQ, HA1-tagged aequorin; PCR, polymerase chain reaction. (1Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Crossref PubMed Scopus (422) Google Scholar). Although microinjection of the purified protein in early studies made its use technically challenging, the dynamic range of aequorin has always recommended it for studies of physiological transitions in [Ca2+] i (1Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Crossref PubMed Scopus (422) Google Scholar). The cloning of one of the members of this family of proteins made it possible to transiently transfect cells with cDNAs encoding aequorin and thereby circumvent the need for microinjection (2Inouye S. Noguchi M. Sakaki Y. Takagi Y. Miyata T. Iwanaga S. Miyata T. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3154-3158Crossref PubMed Scopus (294) Google Scholar, 3Charbonneau H. Walsh K.A. McCann R.O. Prendergast F.G. Cormier M.J. Vanaman T.C. Biochemistry. 1985; 24: 6762-6771Crossref PubMed Scopus (94) Google Scholar). Recently, aequorin has been creatively applied to the measurement of Ca2+ in discrete cellular subdomains, such as the near mitochondrial membrane (4Rizzuto R. Simpson A.W. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (778) Google Scholar, 5Rizzuto R. Bastianutto C. Brini M. Murgia M. Pozzan T. J. Cell Biol. 1994; 126: 1183-1194Crossref PubMed Scopus (308) Google Scholar, 6Rizzuto R. Brini M. Pozzan T. Methods Cell Biol. 1994; 40: 339-358Crossref PubMed Scopus (63) Google Scholar) and the endoplasmic reticulum (7Kendall J.M. Badminton M.N. Dormer R.L. Campbell A.K. Anal. Biochem. 1994; 221: 173-181Crossref PubMed Scopus (56) Google Scholar, 8Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (262) Google Scholar), by creating chimeras of targeting sequences of intracellular organelle marker proteins with aequorin. This offers a clear advantage of selective measurement of [Ca2+] i in various subcellular domains over the current alternative of fluorescent dyes that are distributed uniformly in the cytosol.Ca2+-sensitive adenylyl cyclases (even when transfected heterologously) are directly regulated by changes in [Ca2+] i (9Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar, 10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar). The regulation by Ca2+ is due predominantly to capacitative Ca2+entry; Ca2+ released from internal stores or nonspecific entry of Ca2+ via ionophore is unable to regulate Ca2+-sensitive adenylyl cyclases (11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, somewhat paradoxical is the fact that measurements of global cytosolic [Ca2+] i, using the fluorescent Ca2+-indicator fura-2, reveal that Ca2+released from internal stores or nonspecific entry of Ca2+via ionophore reports far higher levels of [Ca2+] i than are achieved by capacitative Ca2+ entry (11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Such findings have led us to propose that Ca2+-sensitive adenylyl cyclases may be in close proximity to capacitative channels and that the actual [Ca2+] i that the enzyme encounters may be significantly different from cytosolic [Ca2+] (12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Therefore, in the present study, we have constructed a full-length chimera of aequorin and the Ca2+-inhibitable type VI adenylyl cyclase (ACVI) with the purpose of measuring [Ca2+] i, under a variety of physiological conditions, in the microdomain in which Ca2+-sensitive adenylyl cyclases reside.RESULTSThe design chosen for the adenylyl cyclase/aequorin chimera was to place aequorin, linked at the N terminus with the HA1-epitope, downstream of the murine ACVI. It seemed that the adenylyl cyclase might tolerate the inclusion of a large addition at the C-terminal, since the Drosophila ACI homolog carries a substantial C-terminal addition with no deleterious consequence for its activity (27Levin L.R. Han P.L. Hwang P.M. Feinstein P.G. Davis R.L. Reed R.R. Cell. 1992; 68: 479-491Abstract Full Text PDF PubMed Scopus (380) Google Scholar). In addition, substitution at the N terminus of aequorin does not affect its activity, whereas substitution at the C terminus eliminates Ca2+ binding (28Nomura M. Inouye S. Ohmiya Y. Tsuji F.I. FEBS Lett. 1991; 295: 63-66Crossref PubMed Scopus (61) Google Scholar). The strategy used to construct the full-length ACVI-aequorin chimera was first to adopt a two-stage PCR protocol to ligate the 3′ 356 base pairs of the ACVI (13Yoshimura M. Cooper D.M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (201) Google Scholar) upstream of, and in frame with, HA1-aequorin (25Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). This initial construct could then be readily ligated to the major 5′ component of the ACVI cDNA. After confirming the desired sequence of the cloned PCR fusion product, the partial cyclase/aequorin chimera was religated to the remainder of the ACVI cDNA (Fig. 1), and the full-length ACVI/AEQ cDNA was expressed in HEK 293 cells.Since the chimera included the full coding sequence of an adenylyl cyclase, it was possible to determine whether a functional adenylyl cyclase activity was expressed and to evaluate the subcellular localization of any such activity. Thus, vector alone (control), ACVI, and ACVI/AEQ were transfected into HEK 293 cells. Cells were lysed and fractionated on a continuous sucrose gradient (Fig. 2), and adenylyl cyclase and marker enzyme activities were assessed. Both wild type- and ACVI/AEQ-transfected cells showed exactly the same sedimentation profile as control-transfected cells, although the adenylyl cyclase-transfected cells had 3-fold higher activities than control-transfected cells (Fig. 2). Adenylyl cyclase activity sedimented with 5′-nucleotidase activity (an enzyme known to reside at the plasma membrane) and at a lighter density than the mitochondrial fraction (indicated by cytochrome c oxidase activity), which supports its appropriate plasma membrane localization.The foregoing biochemical fractionation data indicate appropriate subcellular localization of the adenylyl cyclase chimera by gross criteria and apparently unchanged overall activity. 2Immunohistochemical experiments using a pan-cyclase antibody directed against the C terminus indicated that approximately 25% of HEK 293 cells transfected with either wild type or chimeric ACVI cDNA showed strong immunoreactivity at the cell periphery consistent with plasma membrane labeling (N. Mons and D. M. F. Cooper, unpublished observations). The next series of experiments asked whether the full functional properties associated with ACVI were retained in the chimera. ACVI is an adenylyl cyclase that is inhibited by [Ca2+] in the submicromolar range in vitroand inhibited strictly by capacitative Ca2+ entry in vivo (11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13Yoshimura M. Cooper D.M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6716-6720Crossref PubMed Scopus (201) Google Scholar, 24Boyajian C.L. Garritsen A. Cooper D.M.F. J. Biol. Chem. 1991; 266: 4995-5003Abstract Full Text PDF PubMed Google Scholar). The fractions corresponding to the peak adenylyl cyclase activity from sucrose gradients (viz. 32–44% sucrose; see Fig. 2) were pooled and sedimented, and their response to a range of EGTA-buffered Ca2+ concentrations was evaluated. When the adenylyl cyclase activity in plasma membranes from either the ACVI- or the ACVI/AEQ-transfected HEK 293 cells are compared, it is clear that a significant (approximately 3-fold) increment in activity is obtained over that of cells transfected with vector alone (Fig. 3 A). Strikingly, the chimeric adenylyl cyclase shows exactly the same concentration dependence for inhibition by Ca2+ as the unmodified ACVI. Maximal inhibition of approximately 40% is observed at 1 μm Ca2+. As an internal control for the free [Ca2+] established in this assay, cerebral cortical membrane adenylyl cyclase activity was assayed and yielded a stimulatory response over the same range of free [Ca2+], in keeping with the predominance of Ca2+-stimulated isoforms of adenylyl cyclase in this tissue (9Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar, 10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar).Figure 3Ca2+ inhibition of ACVI and ACVI/AEQ cyclase activity. A, inhibition by defined pCa2+ of ACVI and ACVI/AEQ in HEK 293 cell membranes. Membranes prepared from HEK 293 cells transiently transfected with ACVI (•), ACVI/AEQ (▪) or the vector alone (○) were assayed in the presence of prostaglandin E1 (10 μm), forskolin (20 μm), and a range of free [Ca2+]. Membrane preparations of mouse cerebral cortex, which express predominantly Ca2+/calmodulin-stimulable adenylyl cyclase activity were assayed in the same conditions, with the addition of calmodulin (1 μm; ▵), to corroborate the specificity of the effects produced by submicromolar [Ca2+] on these two forms of adenylyl cyclase. Data are means ± S.E. of triplicate determinations from an experiment that was repeated twice with similar results. B, effect of capacitative Ca2+ entry on the activity of expressed ACVI and ACVI/AEQ in HEK 293 cells. cAMP accumulation was measured in intact HEK 293 cells transiently expressing vector alone (○), ACVI (•), or ACVI/AEQ (▪) and plotted as a function (percentage) of the Ca2+-free condition. (Values are normalized from a control cAMP accumulation of 1.0, ACVI/AEQ activity was 1.5, and ACVI activity was 3.5 in the experiment shown.) Capacitative Ca2+ entry was evoked by emptying the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores with the sarco-/endoplasmic reticulum calcium ATPase inhibitor thapsigargin (100 nm) in a Ca2+-free Krebs medium. After 4 min, the indicated [Ca2+]ex were added to promote Ca2+ influx, and cAMP accumulation was measured over the subsequent minute. Assays were conducted in the presence of forskolin (10 μm) and prostaglandin E1 (20 μm), which stimulates adenylyl cyclase via the G-protein αs subunit. Data are means ± S.D. of triplicate determinations from an experiment that was repeated four times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The critical biological hallmark of Ca2+-sensitive adenylyl cyclases, whether they are stimulated or inhibited by [Ca2+] i, is a strict dependence on capacitative Ca2+ entry for their regulation in vivo, rather than release from calcium stores or nonspecific cytosolic elevation of [Ca2+] i (9Cooper D.M.F. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (553) Google Scholar, 10Mons N. Cooper D.M.F. Trends Neurosci. 1995; 18: 536-542Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Therefore, the most discriminating test of the appropriate cellular localization of the chimeric construct was whether it retained sensitivity to capacitative Ca2+ entry. Thus, the sensitivity to capacitative Ca2+ entry of the chimeric construct and the native ACVI was compared (Fig. 3 B). HEK 293 cells transfected with control vector, ACVI, or ACVI/AEQ were treated with the sarco-/endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, in the absence of extracellular Ca2+ to deplete inositol 1,4,5-trisphosphate-sensitive internal stores and to activate capacitative Ca2+ entry mechanisms. Such a regimen has been shown previously to generate robust capacitative Ca2+entry, which in turn regulates both endogenous and transfected Ca2+-sensitive adenylyl cyclases (11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). When a range of extracellular [Ca2+] were introduced to the bathing medium to initiate capacitative Ca2+ entry and the cAMP response measured, it was clear that both ACVI- and ACVI/AEQ-transfected cells showed the same (approximately 40%) maximal inhibition (Fig. 3 B). In the presence of 4 mmCa2+, the activity of ACVI and ACVI/AEQ was reduced to 61 ± 7.9% and 58 ± 4%, respectively (Fig. 3 B). These values were not statistically different from each other but were quite clearly statistically different from controls, p< 0.005. These data then unequivocally establish the cellular disposition of the chimeric adenylyl cyclase and predict its probable ability to serve as a sensor of Ca2+ at the plasma membrane.The final series of experiments evaluated the ability of the adenylyl cyclase/aequorin chimera to measure [Ca2+], both in vitro and in vivo compared with cytAEQ. An in vitro calibration curve of cytAEQ and ACVI/AEQ luminescence was fitted to EGTA-determined free [Ca2+] (Fig.4). After a crude separation of cell lysate from the cell membrane fraction for use in the in vitro calibration assay, 89% of the cytAEQ luminescence activity was in the cell lysate, while 89% of the ACVI/AEQ luminescence activity was in the cell membrane fraction. The results shown in Fig. 4 demonstrate that the aequorin portion of the ACVI/AEQ chimera is active and can measure a range of [Ca2+]free in vitro. The relationship between [Ca2+] and log(L/L max) for ACVI/AEQ was not significantly different from that of cytAEQ. Together, these data indicate that fusing the type VI adenylyl cyclase to the N terminus of aequorin does not significantly alter its Ca2+-sensing activity. Nonlinear curve fitting of the untransformed data yielded macroscopic K d values of 1.71 × 10−5m and 1.78 × 10−5m and Hill coefficients of 2.38 and 2.45 for cytAEQ and ACVI/AEQ, respectively.Figure 4Measurement of [Ca2+] using the aequorin-mediated luminescence activity of ACVI/AEQ and cytAEQ. In vitro calibration of ACVI/AEQ and cytAEQ luminescence to EGTA-buffered [Ca2+] was performed. TheL/L max ratios were obtained by determining the immediate rate of light output (L, counts/s) upon mixing cell lysates from cytAEQ-expressing cells or cell membranes from ACVI/AEQ-expressing cells with known [Ca2+] and determining the total integrated number of counts in the sample (L max) upon exposure to saturating [Ca2+]. Data are expressed as the mean ± S.E. of triplicate determinations from an experiment that was repeated twice with similar results. (The in vitro[Ca2+]/log(L/L max) relationships for ACVI/AEQ and cytAEQ were virtually identical in two separate experiments.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether the plasma membrane-targeted ACVI/AEQ could report changes in [Ca2+] i in vivo, the luminescence of ACVI/AEQ and cytAEQ were evaluated in transfected HEK 293 cells under conditions of agonist-induced Ca2+-release from intracellular stores or capacitative Ca2+ entry (Fig.5). The addition of the muscarinic cholinergic agonist, carbachol, to a suspension of HEK 293 cells transfected with either the cytAEQ or the ACVI/AEQ in the absence of extracellular Ca2+produced a rapid rise (<3 s to peak) in aequorin luminescence, which was converted to [Ca2+] i (Fig. 5 A). Luminescence did not increase in response to carbachol in cells where the intracellular Ca2+ stores had been depleted by a pretreatment with 200 nm thapsigargin, which demonstrated that the aequorin-expressing cells were actually measuring increases in [Ca2+] i due to Ca2+ release (data not shown; these conditions mimic earlier treatments of these cells using fura-2 to measure [Ca2+] i; Ref. 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Cells transfected with the cytosolically distributed cytAEQ reported a significantly larger peak in [Ca2+] i upon stimulation by 200 μm carbachol than cells expressing the plasma membrane-bound ACVI/AEQ: 2105 ± 364 nm versus 1160 ± 136 nm (n = 3), respectively (Fig. 5 A). The likely explanation for this difference is the disparate location of the two Ca2+sensors within the cell, since there was no difference in their ability to measure Ca2+ in vitro (Fig.4). 3It is important to note that these differences cannot reflect different degrees of expression of the two probes, since an L max value is measured for each determination to normalize differences in expression. Furthermore, expression of the cyclase chimera (or other Ca2+-sensitive adenylyl cyclases) does not affect global [Ca2+] i rises, as measured by fura-2 Ca2+ (data not shown). The plasma membrane-bound Ca2+-sensor (ACVI/AEQ) detects Ca2+, which is released from intracellular stores at a level that is considerably lower than the levels detected by the uniformly cytosolically distributed Ca2+-sensor (cytAEQ), presumably due to the reuptake into intracellular Ca2+ stores and the activation of Ca2+ extrusion mechanisms. If this explanation is correct, it would be predicted that the plasma membrane-bound ACVI/AEQ would detect higher [Ca2+] i during Ca2+ entry than the cytosolic aequorin. To evaluate this prediction, capacitative Ca2+ entry was activated in HEK 293 cells transfected with either the ACVI/AEQ or the cytAEQ by adding 4 mm Ca2+ to cells that had been pretreated with 100 nm thapsigargin for 10 min in nominally Ca2+-free buffer (Fig. 5 B). In support of the prediction, cells expressing the plasma membrane-bound ACVI/AEQ reported a rapid rise in [Ca2+] i, which was consistently higher than was detected by the cytosolically distributed cytAEQ: 1225 ± 117 nm versus 660 ± 61 nm (n = 5), respectively.Figure 5In vivo [Ca2+] i measurements with cytAEQ- and ACVI/AEQ-transfected HEK 293 cells. A, HEK 293 cells transfected with either cytAEQ or ACVI/AEQ were stimulated with carbachol (200 μm) in Ca2+-free buffer (supplemented with 0.2 mmEGTA) at the time indicated by the arrow, and the resulting luminescence was measured and converted to [Ca2+] i. The upper trace is the carbachol response in cytAEQ cells, and the lower trace is the carbachol response in ACVI/AEQ cells. The traces shown are the means of triplicate determinations from a representative experiment that was repeated on three separate days with similar results.B, HEK 293 cells transfected with either cytAEQ or ACVI/AEQ were pretreated with 100 nm thapsigargin for 10 min in Ca2+-free buffer to activate capacitative Ca2+channels. Prior to the start of the experiment, 0.2 mm EGTA was added, and capacitative Ca2+ entry was initiated by adding 4 mm Ca2+ at the times indicated by thearrows. The resulting luminescence was measured and converted to [Ca2+] i. The upper traceis the capacitative Ca2+ entry response in the ACVI/AEQ cells, and the lower trace is the capacitative Ca2+ entry response in the cytAEQ cells. The traces shown are the means of triplicate determinations from a representative experiment that was repeated on 5 separate days with similar results. All experiments were performed at 30 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONBy a wide variety of criteria, the presently described adenylyl cyclase/aequorin chimera is fully functional and retains the properties of both parent molecules. The adenylyl cyclase/aequorin is localized in the same cellular regions as native ACVI; the activities of the two forms of the enzyme and their sensitivity to Ca2+, bothin vitro and in vivo, are identical. Aequorin, whether free in the cytosol or tagged onto the C terminus of ACVI, has unaltered affinities and properties for responding to Ca2+and can measure [Ca2+] i. In the intact cell, the ACVI/AEQ reports lower concentrations of released Ca2+reaching the plasma membrane than does the cytosolically distributed cytAEQ. During capacitative Ca2+ entry, however, the ACVI/AEQ reports a much higher [Ca2+] i at the plasma membrane than is seen by the cytosolically distributed cytAEQ. This finding is quite compatible with the reuptake, buffering, and extrusion mechanisms that are expected to reduce the amount of released Ca2+ reaching microdomains within the plasma membrane and to reduce the amount of Ca2+ reaching the cytosol during Ca2+ influx. These data provide an explanation for the fact that capacitative influx preferentially regulates Ca2+-sensitive adenylyl cyclases rather than Ca2+ released from internal stores, although global increases in [Ca2+] i due to released Ca2+ are much greater (11Chiono M. Mahey R. Tate G. Cooper D.M.F. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12Fagan K.A. Mahey R. Cooper D.M.F. J. Biol. Chem. 1996; 271: 12438-12444Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar).A wide variety of precise information can be anticipated from the application of this construct to a range of physiological situations. For instance, the dynamics of [Ca2+] i in the immediate environment of a Ca2+-sensitive adenylyl cyclase and how this relates to the regulation of the enzyme can be determined. This is impossible to do with a cytosolically distributed Ca2+ indicator. Along with the limitation of not being able to resolve Ca2+ dynamics within a microdomain, cytosolically distributed fluorescent EGTA derivatives such as fura-2 can reach intracellular concentrations of 20 μm and greater. At these concentrations, fluorescent Ca2+-indicators such as fura-2 can actually alter the true kinetics of the response by acting as a Ca2+ buffer (25Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). However, the concentration of a recombinantly expressed protein would be considerably lower than 1 μm and would not be expected to contribute to the intracellular Ca2+ buffering capacity. In addition, the cyclase chimera can serve as a novel plasma membrane-embedded [Ca2+] i sensor, which, given the extreme dependence of Ca2+-sensitive cyclases on capacitative entry rather than release, could allow the monitoring of a very selective subpool of [Ca2+] i. The jellyfish protein aequorin with its co-factor, coelenterazine, has been used for over 3 decades to measure cytosolic calcium ([Ca2+] i) 1The abbreviations used are: [Ca2+] i, cytosolic calcium; HEK 293 cells, human embryonic kidney cells; ACVI, adenylyl cyclase type VI; ACVI/AEQ, adenylyl cyclase/aequorin chimera; cytAEQ, HA1-tagged aequorin; PCR, polymerase chain reaction. 1The abbreviations used are: [Ca2+] i, cytosolic calcium; HEK 293 cells, human embryonic kidney cells; ACVI, adenylyl cyclase type VI; ACVI/AEQ, adenylyl cyclase/aequorin chimera; cytAEQ, HA1-tagged aequorin; PCR, polymerase chain reaction. (1Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Crossref PubMed Scopus (422) Google Scholar). Although microinjection of the purified protein in early studies made its use technically challenging, the dynamic range of aequorin has always recommended it for studies of physiological transitions in [Ca2+] i (1Cobbold P.H. Rink T.J. Biochem. J. 1987; 248: 313-328Crossref PubMed Scopus (422) Google Scholar). The cloning of one of the members of this family of proteins made it possible to transiently transfect cells with cDNAs encoding aequorin and thereby circumvent the need for microinjection (2Inouye S. Noguchi M. Sakaki Y. Takagi Y. Miyata T. Iwanaga S. Miyata T. Tsuji F.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3154-3158Crossref PubMed Scopus (294) Google Scholar, 3Charbonneau H. Walsh K.A. McCann R.O. Prendergast F.G. Cormier M.J. Vanaman T.C. Biochemistry. 1985; 24: 6762-6771Crossref PubMed Scopus (94) Google Scholar). Recently, aequorin has been creatively applied to the measurement of Ca2+ in discrete cellular subdomains, such as the near mitochondrial membrane (4Rizzuto R. Simpson A.W. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (778) Google Scholar, 5Rizzuto R. Bastianutto C. Brini M. Murgia M. Pozzan T. J. Cell Biol. 1994; 126: 1183-1194Crossref PubMed Scopus (308) Google Scholar, 6Rizzuto R. Brini M. Pozzan T. Methods Cell Biol. 1994; 40: 339-358Crossref PubMed Scopus (63) Google Scholar) and the endoplasmic reticulum (7Kendall J.M. Badminton M.N. Dormer R.L. Campbell A.K. Anal. Biochem. 1994; 221: 173-181Crossref PubMed Scopus (56) Google Scholar, 8Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (262) Google Scho" @default.
• W2072484997 created "2016-06-24" @default.
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• W2072484997 creator A5071171292 @default.
• W2072484997 creator A5076601399 @default.
• W2072484997 creator A5077317216 @default.
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• W2072484997 date "1997-07-01" @default.
• W2072484997 modified "2023-09-26" @default.
• W2072484997 title "Construction of a Full-length Ca2+-sensitive Adenylyl Cyclase/Aequorin Chimera" @default.
• W2072484997 cites W146759131 @default.
• W2072484997 cites W1771946992 @default.
• W2072484997 cites W1847257428 @default.
• W2072484997 cites W1939705865 @default.
• W2072484997 cites W1967513994 @default.
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