Functional Co-localization of Transfected Ca2+-stimulable Adenylyl Cyclases with Capacitative Ca2+ Entry Sites

Fagan, Kent A.; Mahey, Rajesh; Cooper, Dermot M.F.

doi:10.1074/jbc.271.21.12438

Cited by 158 publications

(198 citation statements)

References 34 publications

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“…A central issue in implicating the Ca 2ϩ responsiveness of an adenylyl cyclase in a physiological process concerns the correspondence between the concentrations of Ca 2ϩ achieved in response to physiological stimuli versus those required to regulate these adenylyl cyclases in vitro. 1 This issue seems moot with Ca 2ϩ -stimulable adenylyl cyclases, which are stimulated by Ca 2ϩ in vitro and are also stimulated in response to various physiological means of elevating Ca 2ϩ in vivo (2,7,9). Such correlations are mutually supportive.…”

Section: ؉mentioning

confidence: 79%

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Inhibition by Calcium of Mammalian Adenylyl Cyclases

Guillou

Nakata

Cooper

1999

Journal of Biological Chemistry

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108

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Ca2؉ regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca 2؉ inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca 2؉ -stimulable and Ca 2؉ -inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca 2؉ responses, suggesting the presence of two Ca 2؉ sites of high (ϳ0.2 M) and low affinity (ϳ0.1 mM). With a high affinity, Ca 2؉ (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca 2؉ inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg 2؉ site(s). A remarkable selectivity for Ca 2؉ was displayed by the high affinity site, with a K i value of ϳ0.2 M, in the face of a 5000-fold excess of Mg 2؉. The present results show that high and low affinity inhibition by Ca 2؉ can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca 2؉ -inhibitable adenylyl cyclases.Profound physiological significance derives from the regulation of adenylyl cyclase by Ca 2ϩ

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Section: ؉mentioning

confidence: 79%

“…Indeed, in membranes from transfected HEK 293 cells, AC1 and AC8 were stimulated by submicromolar concentrations of Ca 2ϩ , while they displayed inhibition in response to higher concentrations, mirroring what happens in most brain tissues (9). However, again, the situation with the Ca 2ϩ -inhibitable adenylyl cyclases is less clear.…”

Section: ؉mentioning

confidence: 95%

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Guillou

Nakata

Cooper

1999

Journal of Biological Chemistry

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108

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“…Adenyl cyclase AC-1 and -8 isoforms are apically expressed in airway epithelial cells in culture (27) and are stimulated by Ca 2+ in a calmodulin-dependent manner (28). AC-1 and AC-8 are insensitive to Ca 2+ release from intracellular stores, and are rather stimulated by Ca 2+ entry (29). α7 nAChR is characterized by an elevated Ca 2+ permeability (15).…”

Section: Resultsmentioning

confidence: 99%

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Maouche

Medjber

Zahm

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na + absorption, a defective cAMP-dependent Cl − conductance, higher concentrations of Na + , Cl − , K + , and Ca 2+ in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca 2+ entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.chloride efflux | ciliated cell | mouse | mucociliary clearance | submucosal gland

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“…In nonexcitable cells, this regulation by [Ca 2ϩ ] i is orchestrated by a strict dependence on the entry of Ca 2ϩ that is triggered by the emptying of internal Ca 2ϩ stores, the so-called capacitative Ca 2ϩ entry (CCE) (2). Other means of elevating [Ca 2ϩ ] i , such as release from stores or ionophore-mediated entry, are ineffective (3)(4)(5). The dependence of Ca 2ϩ -sensitive adenylyl cyclases on regulation by CCE led to the suggestion that adenylyl cyclases and Ca 2ϩ entry channels were functionally colocalized (5).…”

mentioning

confidence: 99%

Residence of Adenylyl Cyclase Type 8 in Caveolae Is Necessary but Not Sufficient for Regulation by Capacitative Ca2+Entry

Smith

Fagan

et al. 2002

Journal of Biological Chemistry

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107

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(2000) J. Biol. Chem. 275, 26530 -26537). In the present study, we established that depletion of cellular cholesterol ablated the regulation by CCE of a Ca 2؉ -stimulable adenylyl cyclase, AC8, heterologously expressed in HEK293 cells. We considered the possibility that a calmodulin-binding domain in the N terminus of AC8, which is not required for in vitro regulation by Ca 2؉ , might play a targeting role. Deletion and mutation of the N terminus did attenuate the enzyme's sensitivity to CCE without altering its in vitro responsiveness to Ca 2؉ /calmodulin. Both N terminus-deleted AC8 and wild type AC8 were expressed at the plasma membrane, as shown by imaging analysis of green fluorescence protein-tagged constructs. However, not only wild type AC8 but also the CCE-insensitive mutants occurred in caveolar fractions of the plasma membranes, even though a Ca 2؉ -insensitive adenylyl cyclase, AC7, was excluded from caveolae. Finally, the AC8 mutants were no more responsive to nonphysiological elevation of Ca 2؉ than the wild type. We conclude that (i) not all adenylyl cyclases reside in caveolae, (ii) the calmodulin-binding domain in the N terminus of AC8 does not play a role in caveolar targeting, (iii) the N terminus does play a role in associating AC8 with factors that confer sensitivity to CCE, and (iv) residence of Ca 2؉ -sensitive adenylyl cyclases in caveolae is essential but not sufficient for regulation by CCE.

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Functional Co-localization of Transfected Ca2+-stimulable Adenylyl Cyclases with Capacitative Ca2+ Entry Sites

Cited by 158 publications

References 34 publications

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Residence of Adenylyl Cyclase Type 8 in Caveolae Is Necessary but Not Sufficient for Regulation by Capacitative Ca2+Entry

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