Casey Balslev posted an update 3 months ago
6 ) and similar kinetic properties of the wild-type and F163A mutant PDE5 ( Fig. Point mutations (F778A and G617P) altered basal BRET of the PDE5 sensor ( Figs. Metal ion binding to the catalytic domain of PDE5 did not cause a major change in BRET ( Fig.
Importantly, binding of Ca2+ to the catalytic domain, while not permitting catalysis, could provide an added means of PDE5 regulation, especially in the contracted state of smooth muscle cells ( 54 ) where high intracellular Ca2+ levels are seen. Mg2+ is known to regulate a number of cellular processes, and its intracellular free levels are tightly regulated ( 50 , 51 ). Therefore, hormones and vasoactive agents ( 52 ) that regulate the free intracellular levels of Mg2+ could also alter PDE5 activity. However, the high entropic contribution seen in the case of cGMP could arise from the selection of conformations” with stabilized helix α4 and β2-β3 loop (including the short helix α2/3) ( 35 ) and altered the relative positioning and/or orientation of helices α2 and α5 ( supplemental Fig.
8 C). No considerable BRET change was observed in the absence of ligand at different temperatures, and the gross structural integrity of the PDE5 sensor in this temperature range was ascertained by similar luciferase activities at all the temperatures tested ( supplemental Fig. We then extended our studies to suggest the distinct thermodynamic bases for cGMP- and Fildena-induced conformational changes. Plot of the ln of the ratio of change in BRET in the presence of cGMP or Fildena to basal BRET versus 1/T (K−1) is shown.
8 A). Incubation of the D612A mutant sensor with MgCl2 did not alter the EC50 of the Fildena-induced conformational change ( Fig. We incubated lysates with varying concentrations of Fildena in the presence of 10 mm MgCl2 and observed a striking decrease in the EC50 value (EC50 29.1 ± 14 pm; Fig. Given the allosteric effect of metal ions on the cGMP-induced structural change, we proceeded to investigate the effect of metal ions on Fildena-induced structural change.
To demonstrate unequivocally that the presence of metal ions in cells modulates the conformation of PDE5, we reduced intracellular metal ion concentrations by incubating cells in media lacking either MgCl2, CaCl2 or both. C, cells expressing the wild-type or D612A mutant PDE5 sensor were incubated in Hanks’ balanced salt solution and the indicated metals (as chloride salts) for 1 h. After incubation, cells were treated with 1 mm DETA/NO for 5 min, and percentage decreases in BRET are plotted. B, cells expressing wild-type, D612A, or F163A/D612A mutant PDE5 sensors were treated with 1 mm DETA/NO for 5 min, and the percentage decrease in the BRET is plotted.
Catalytic metal ion binding prevents cGMP-induced conformational change in PDE5. Therefore, metal ion binding at the catalytic site modified the conformational change induced on cGMP binding to the GAFa domain in PDE5.