Parameters Used to Simulate Transient cAMP Signals Near the Plasma Membrane of HEK-293 Cells
| Parameter . | Parameter definition . | Value . | Initial condition . | Reference . |
|---|---|---|---|---|
| [βtot] | Total β2AR concentration | 1 μM | ** | |
| [β] | Inactive receptor concentration | 0.2 μM | ||
| [β*] | Activated receptor concentration | 0.8 μM | ||
| [βdesense] | Desensitized receptor concentration | 0 μM | ||
| [βint] | Internalized receptor concentration | 0 μM | ||
| kGRK | Rate constant of receptor phosphorylation | 0.005 s−1 | Tran et al., 2004 | |
| krpp | Rate constant of receptor dephosphorylation | 0.0005 s−1 | Iyer et al., 2006; Tran et al., 2007a | |
| kint | Rate constant of receptor internalization | 0.01 s−1 | ||
| krec | Rate constant of receptor recycling | 0.005 s−1 | ||
| kact | Rate constant of Gs activation of β2AR | 15 s−1 | Frace et al., 1993 | |
| kh | Rate constant of GTP hydrolysis | 0.8 s−1 | Frace et al., 1993 | |
| KD | Dissociation constant of isoproterenol | 250 nM | Whaley et al., 1994 | |
| Gstot | Total concentration of Gs | 4 μM | ** with initial estimate from Post et al., 1995 | |
| Kc | Equilibrium constant between activated receptor and G protein | 15 μM | ||
| KGsAC | Equilibrium constant between activated G protein and AC | 315 μM | ||
| ACsyn | cAMP synthesis rate | 10 μM·s−1 | ** | |
| AC0 | Basal cAMP synthesis rate | 0.005 μM·s−1 | ** | |
| [N] | cAMP concentration | 0.5 μM | Beavo et al., 1974 | |
| Vmax-PDE | Maximal hydrolysis rate of unphosphorylated PDE | 0.15 μM⋅s−1 | Reeves et al., 1987; Rich et al., 2001a, 2007 | |
| Vmax-PDEp | Maximal hydrolysis rate of phosphorylated PDE | 2.5⋅ Vmax-PDE | Rich et al., 2007 | |
| Km | Michaelis constant for PDE | 1 μM | Houslay et al., 1998 | |
| KI | Inhibition constant of rolipram | 0.1 μM | Houslay et al., 1998; Richter and Conti, 2004 | |
| K1/2 | cAMP concentration that half maximally activates CNG channels | 1.1 μM | Rich et al., 2001a,b | |
| N | Hill coefficient of cAMP binding to CNG channels | 2.1 | Dhallan et al., 1990; Rich et al., 2001b | |
| kPKA | Rate constant of PKA-mediated phosphorylation | 0.015 μM−1·s−1 | ** | |
| kpp | Rate constant of PDE dephosphorylation | 0.005 s−1 | ** | |
| [PKA] | PKA concentration | 1 μM | Beavo et al., 1974; Hofmann et al., 1977; Rich and Karpen, 2002 | |
| kfa | Rate constant of cAMP binding to site a of the R subunit | 5 μM−1·s−1 | Doskeland and Ogreid, 1984; Rich and Karpen, 2002 | |
| kfb | Rate constant of cAMP binding to site b | 0.4 μM−1·s−1 | Doskeland and Ogreid, 1984; Rich and Karpen, 2002 | |
| kra | Rate constant of cAMP dissociation from site a | 1 s−1 | Doskeland and Ogreid, 1984; Houge et al., 1990; Rich and Karpen, 2002 | |
| krb | Rate constant of cAMP dissociation from site b | 0.2 s−1 | Doskeland and Ogreid, 1984; Houge et al., 1990; Rich and Karpen, 2002 | |
| kPKAact | Rate constant of R and C subunit dissociation | 70 s−1 | Smith et al., 1981; Houge et al., 1990; Huang and Taylor, 1998; Rich and Karpen, 2002 | |
| kPKAdeact | Rate constant of R and C subunit association | 0.75 μM−2·s−1 | Smith et al., 1981; Houge et al., 1990; Huang and Taylor, 1998; Rich and Karpen, 2002 | |
| KI-PKI | Inhibition constant of PKI | 2.3 nM | Cheng et al., 1986 |
| Parameter . | Parameter definition . | Value . | Initial condition . | Reference . |
|---|---|---|---|---|
| [βtot] | Total β2AR concentration | 1 μM | ** | |
| [β] | Inactive receptor concentration | 0.2 μM | ||
| [β*] | Activated receptor concentration | 0.8 μM | ||
| [βdesense] | Desensitized receptor concentration | 0 μM | ||
| [βint] | Internalized receptor concentration | 0 μM | ||
| kGRK | Rate constant of receptor phosphorylation | 0.005 s−1 | Tran et al., 2004 | |
| krpp | Rate constant of receptor dephosphorylation | 0.0005 s−1 | Iyer et al., 2006; Tran et al., 2007a | |
| kint | Rate constant of receptor internalization | 0.01 s−1 | ||
| krec | Rate constant of receptor recycling | 0.005 s−1 | ||
| kact | Rate constant of Gs activation of β2AR | 15 s−1 | Frace et al., 1993 | |
| kh | Rate constant of GTP hydrolysis | 0.8 s−1 | Frace et al., 1993 | |
| KD | Dissociation constant of isoproterenol | 250 nM | Whaley et al., 1994 | |
| Gstot | Total concentration of Gs | 4 μM | ** with initial estimate from Post et al., 1995 | |
| Kc | Equilibrium constant between activated receptor and G protein | 15 μM | ||
| KGsAC | Equilibrium constant between activated G protein and AC | 315 μM | ||
| ACsyn | cAMP synthesis rate | 10 μM·s−1 | ** | |
| AC0 | Basal cAMP synthesis rate | 0.005 μM·s−1 | ** | |
| [N] | cAMP concentration | 0.5 μM | Beavo et al., 1974 | |
| Vmax-PDE | Maximal hydrolysis rate of unphosphorylated PDE | 0.15 μM⋅s−1 | Reeves et al., 1987; Rich et al., 2001a, 2007 | |
| Vmax-PDEp | Maximal hydrolysis rate of phosphorylated PDE | 2.5⋅ Vmax-PDE | Rich et al., 2007 | |
| Km | Michaelis constant for PDE | 1 μM | Houslay et al., 1998 | |
| KI | Inhibition constant of rolipram | 0.1 μM | Houslay et al., 1998; Richter and Conti, 2004 | |
| K1/2 | cAMP concentration that half maximally activates CNG channels | 1.1 μM | Rich et al., 2001a,b | |
| N | Hill coefficient of cAMP binding to CNG channels | 2.1 | Dhallan et al., 1990; Rich et al., 2001b | |
| kPKA | Rate constant of PKA-mediated phosphorylation | 0.015 μM−1·s−1 | ** | |
| kpp | Rate constant of PDE dephosphorylation | 0.005 s−1 | ** | |
| [PKA] | PKA concentration | 1 μM | Beavo et al., 1974; Hofmann et al., 1977; Rich and Karpen, 2002 | |
| kfa | Rate constant of cAMP binding to site a of the R subunit | 5 μM−1·s−1 | Doskeland and Ogreid, 1984; Rich and Karpen, 2002 | |
| kfb | Rate constant of cAMP binding to site b | 0.4 μM−1·s−1 | Doskeland and Ogreid, 1984; Rich and Karpen, 2002 | |
| kra | Rate constant of cAMP dissociation from site a | 1 s−1 | Doskeland and Ogreid, 1984; Houge et al., 1990; Rich and Karpen, 2002 | |
| krb | Rate constant of cAMP dissociation from site b | 0.2 s−1 | Doskeland and Ogreid, 1984; Houge et al., 1990; Rich and Karpen, 2002 | |
| kPKAact | Rate constant of R and C subunit dissociation | 70 s−1 | Smith et al., 1981; Houge et al., 1990; Huang and Taylor, 1998; Rich and Karpen, 2002 | |
| kPKAdeact | Rate constant of R and C subunit association | 0.75 μM−2·s−1 | Smith et al., 1981; Houge et al., 1990; Huang and Taylor, 1998; Rich and Karpen, 2002 | |
| KI-PKI | Inhibition constant of PKI | 2.3 nM | Cheng et al., 1986 |
Initial conditions were estimated based on steady-state parameter values in the presence of basal adenylyl cyclase activity. ** denotes values that were fit to the time course of cAMP signals as described in Rich et al. (2007). Concentrations of enzymes should be considered approximate, whether estimated or experimentally derived, because, with the exceptions of data provided from our work, experimentally derived data were obtained from other cellular systems, either overexpression systems or other cell types. In addition, there have been few studies estimating the localized concentrations of enzymes within signaling complexes.