Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellul ar functions. Current methods for measuring cAMP lack both temporal and spa tial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP si gnals are simple and contain little information. Here we show the developme nt of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory or subunit responde d rapidly to jumps in cAMP concentration, and their cAMP sensitivity was me asured to calibrate the sensor for intracellular measurements. We used thes e channels to detect cAMP, produced by either heterologously expressed or e ndogenous adenylyl cyclase, in both single cells and cell populations. Afte r forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma ce lls produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk c ytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 2 93 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains , and diffusion of cAMP between these domains and the bulk cytosol is signi ficantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% excha nge in 194 s, compared with 22-56 s for substances that monitor exchange wi th the cytosol),Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth n oting that in models of unrestricted diffusion, even in extreme proximity t o adenylyl cyclase, cAMP does not reach high enough concentrations to subst antially activate PKA or cyclic nucleotide-gated channels, unless the entir e cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signal s should also facilitate the differential regulation of cellular targets.