Abstract
A large number of hormones, neurotransmitters, and odorants exert their effects on cells by triggering changes in intracellular levels of cyclic adenosine monophosphate (cAMP). Although the effector proteins that bind cAMP have been identified, it is not known how this single messenger can differentially regulate the activities of hundreds of cellular proteins. It has been clear, for some time, that compartmentation of cAMP signals must be taking place, but the physical basis for compartmentation and the nature of local cAMP signals are mostly unknown. We present here a high-resolution method for measuring cAMP signals near the membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We outline how these channels can be used in electrophysiological experiments to measure accurately changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We also describe how the method has been employed to dissect the roles of diffusion barriers and differential phosphodiesterase activity in creating distinct cAMP signals. This approach has much greater spatial and temporal resolution than other methods for measuring cAMP and should help to unravel the complexities of signaling by this ubiquitous messenger.
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References
Montminy, M. (1997) Transcriptional regulation by cyclic AMP. Annu. Rev. Biochem. 66, 807–822.
Francis, S. and Corbin, J. D. (1999) Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit. Rev. Clin. Lab. Sci. 36, 275–328.
Finn, J. T., Grunwald, M. E., and Yau, K.-W. (1996) Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu. Rev. Physiol. 58, 395–426.
de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474–477.
Steinberg, S. F. and Brunton, L. L. (2001) Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. 41, 751–773.
Jurevicius, J. and Fischmeister, R. (1996) cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by β-adrenergic agonists. Proc. Natl. Acad. Sci. USA 93, 295–299.
Karpen, J. W. and Rich, T. C. (2001) The fourth dimension in cellular signaling. Science 293, 2204, 2205.
Hall, D. D. and Hell, J. W. (2001) The fourth dimension in cellular signaling—response. Science 293, 2205.
Rich, T. C., Fagan, K. A., Nakata, H., Schaack, J., Cooper, D. M. F., and Karpen, J. W. (2000) Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J. Gen. Physiol. 116, 147–161.
Rich, T. C., Tse, T. E., Rohan, J. G., Schaack, J., and Karpen, J. W. (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J. Gen. Physiol. 118, 63–77.
Rich, T. C., Fagan, K. A., Tse, T. E., Schaack, J., Cooper, D. M. F., and Karpen, J. W. (2001) A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc. Natl. Acad. Sci. USA 98, 13,049–13,054.
Rich, T. C. and Karpen, J. W. (2002) Cyclic AMP sensors in living cells: what signals can they actually measure? Ann. Biomed. Eng. 30, 1088–1099.
Karpen, J. W., Zimmerman, A. L., Stryer, L., and Baylor, D. A. (1988) Gating kinetics of the cyclic-GMP-activated channel of retinal rods: flash photolysis and voltage-jump studies. Proc. Natl. Acad. Sci. USA 85, 1287–1291.
Hagen, V., Dzeja, C., Frings, S., Bendig, J., Krause, E., and Kaupp, U. B. (1996) Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: synthesis and application to cyclic nucleotide-gated channels. Biochemistry 35, 7762–7771.
Sakmann, B. and Neher, E. (1995) Single Channel Recording, Plenum, New York.
Hille, B. (2001) Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA.
Horn, R. and Marty, A. (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145–159.
Pusch, M. and Neher, E. (1988) Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch. 411, 204–211.
Houslay, M. D., Sullivan, M., and Bolger, G. B. (1998) The multienzyme PDE4 cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular targeting, regulation, and selective inhibition by compounds exerting anti-inflammatory and antidepressant actions. Adv. Pharmacol. 44, 225–342.
Stryer, L. (1991) Visual excitation and recovery. J. Biol. Chem. 266, 10,711–10,714.
Yau, K.-W. (1994) Phototransduction mechanism in retinal rods and cones: The Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 35, 9–32.
Molday, R. S. (1998) Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases: The Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 39, 2493–2513.
Kaupp, U. B. and Seifert, R. (2002) Cyclic nucleotide-gated ion channels. Physiol. Rev. 82, 769–824.
Jordan, M., Schallhorn, A., and Wurm, F. M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601.
Rapp, P. E. and Berridge, M. J. (1977) Oscillations in calcium-cyclic AMP control loops form the basis of pacemaker activity and other high frequency biological rhythms. J. Theor. Biol. 66, 497–525.
Cooper, D. M. F., Mons, N., and Karpen, J. W. (1995) Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature 374, 421–424.
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Karpen, J.W., Rich, T.C. (2005). High-Resolution Measurements of Cyclic Adenosine Monophosphate Signals in 3D Microdomains. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:015
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DOI: https://doi.org/10.1385/1-59259-839-0:015
Publisher Name: Humana Press
Print ISBN: 978-1-58829-314-5
Online ISBN: 978-1-59259-839-7
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