Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors

Abstract

Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP] i . Regulatory fragments of PKG I α, PKG I β, and an N-terminal deletion mutant of PKG I α were fused to circular permutated EGFP to generate α-, β-, and δ-FlincG, with high dynamic ranges and apparent K D,cGMP values of 35 nM, 1.1 μM, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. δ-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, δ-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP] i in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, δ-FlincG had an EC 50,cGMP of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC 50,DEA/NO = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP] i , which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets.