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The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

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Amplitude of the microdomain is influenced by cAMP diffusion coefficient, but not by PKAc diffusion coefficient.Impeded diffusion of cAMP is not required for the microdomain, but influences the concentration difference between submembrane and cytosol. The faster the cAMP diffusion coefficient, the smaller the difference between submembrane and cytosol concentration (measured as difference between FRET ΔF/F submembrane and FRET ΔF/F cystosol (solid black line, black squares). cAMP diffusion coefficient ranges from k = 0.5 to k = 3 times its control value of 295 µm2/s). Diffusion of the PKA catalytic subunit plays no significant role in generating cAMP microdomains (solid gray line, open squares). PKAc diffusion constant ranges from k = 0 to k = 2 times its control value of 59.54 µm2/s.
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pone-0011725-g007: Amplitude of the microdomain is influenced by cAMP diffusion coefficient, but not by PKAc diffusion coefficient.Impeded diffusion of cAMP is not required for the microdomain, but influences the concentration difference between submembrane and cytosol. The faster the cAMP diffusion coefficient, the smaller the difference between submembrane and cytosol concentration (measured as difference between FRET ΔF/F submembrane and FRET ΔF/F cystosol (solid black line, black squares). cAMP diffusion coefficient ranges from k = 0.5 to k = 3 times its control value of 295 µm2/s). Diffusion of the PKA catalytic subunit plays no significant role in generating cAMP microdomains (solid gray line, open squares). PKAc diffusion constant ranges from k = 0 to k = 2 times its control value of 59.54 µm2/s.

Mentions: Although these simulations confirm that PDE4s play the main role in controlling cAMP microdomains, diffusion may still play a role because an infinitely fast diffusion constant theoretically would produce a well stirred and homogenous distribution of molecules. To delineate the role of cAMP diffusion and to evaluate the robustness of the model to parameter variations, simulations are repeated with the cAMP diffusion constant ranging from one half to three times its control value, representing the range of experimentally measured values. Simulations show that reducing the speed of cAMP diffusion increases the concentration difference between submembrane and cytosol, while increasing the speed of cAMP diffusion diminishes, but does not eliminate, the cAMP concentration difference (Fig. 7). Thus, the results are not dependent on the precise value chosen for the cAMP diffusion constant. PKA is another important and diffusible molecule in the model; thus, the effect of diffusion of the PKA catalytic subunit (PKAc) also is evaluated, by repeating simulations with the PKAc diffusion constant ranging from one half to two times its control value. Fig. 7 shows that the change in the PKAc diffusion constant produces no change in the magnitude of the cAMP concentration difference, even in the most extreme case with no PKAc diffusion. Though diffusion of the PKA catalytic subunit is slower than cAMP, PKAc diffusion is fast compared to its inactivation (rebinding to the regulatory subunit) so that PKAc diffuses to the cytosol to phosphorylate PDE4D, thereby generating the cAMP microdomain. In summary, the cAMP microdomain does not require impeded diffusion, but the extent of the cAMP concentration difference is affected by the diffusion constant of cAMP, though not that of PKAc.


The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

Amplitude of the microdomain is influenced by cAMP diffusion coefficient, but not by PKAc diffusion coefficient.Impeded diffusion of cAMP is not required for the microdomain, but influences the concentration difference between submembrane and cytosol. The faster the cAMP diffusion coefficient, the smaller the difference between submembrane and cytosol concentration (measured as difference between FRET ΔF/F submembrane and FRET ΔF/F cystosol (solid black line, black squares). cAMP diffusion coefficient ranges from k = 0.5 to k = 3 times its control value of 295 µm2/s). Diffusion of the PKA catalytic subunit plays no significant role in generating cAMP microdomains (solid gray line, open squares). PKAc diffusion constant ranges from k = 0 to k = 2 times its control value of 59.54 µm2/s.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908681&req=5

pone-0011725-g007: Amplitude of the microdomain is influenced by cAMP diffusion coefficient, but not by PKAc diffusion coefficient.Impeded diffusion of cAMP is not required for the microdomain, but influences the concentration difference between submembrane and cytosol. The faster the cAMP diffusion coefficient, the smaller the difference between submembrane and cytosol concentration (measured as difference between FRET ΔF/F submembrane and FRET ΔF/F cystosol (solid black line, black squares). cAMP diffusion coefficient ranges from k = 0.5 to k = 3 times its control value of 295 µm2/s). Diffusion of the PKA catalytic subunit plays no significant role in generating cAMP microdomains (solid gray line, open squares). PKAc diffusion constant ranges from k = 0 to k = 2 times its control value of 59.54 µm2/s.
Mentions: Although these simulations confirm that PDE4s play the main role in controlling cAMP microdomains, diffusion may still play a role because an infinitely fast diffusion constant theoretically would produce a well stirred and homogenous distribution of molecules. To delineate the role of cAMP diffusion and to evaluate the robustness of the model to parameter variations, simulations are repeated with the cAMP diffusion constant ranging from one half to three times its control value, representing the range of experimentally measured values. Simulations show that reducing the speed of cAMP diffusion increases the concentration difference between submembrane and cytosol, while increasing the speed of cAMP diffusion diminishes, but does not eliminate, the cAMP concentration difference (Fig. 7). Thus, the results are not dependent on the precise value chosen for the cAMP diffusion constant. PKA is another important and diffusible molecule in the model; thus, the effect of diffusion of the PKA catalytic subunit (PKAc) also is evaluated, by repeating simulations with the PKAc diffusion constant ranging from one half to two times its control value. Fig. 7 shows that the change in the PKAc diffusion constant produces no change in the magnitude of the cAMP concentration difference, even in the most extreme case with no PKAc diffusion. Though diffusion of the PKA catalytic subunit is slower than cAMP, PKAc diffusion is fast compared to its inactivation (rebinding to the regulatory subunit) so that PKAc diffuses to the cytosol to phosphorylate PDE4D, thereby generating the cAMP microdomain. In summary, the cAMP microdomain does not require impeded diffusion, but the extent of the cAMP concentration difference is affected by the diffusion constant of cAMP, though not that of PKAc.

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

Show MeSH