Cyclic Nucleotide–Gated Channels Colocalize with Adenylyl Cyclase in Regions of Restricted Camp Diffusion

Abstract

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (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 significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with 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 noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire 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 signals should also facilitate the differential regulation of cellular targets.

Cytoplasmic cAMP concentrations in intact cardiac myocytes

“…The size and dimensions of these submembrane compartments are predictions that arise from the ability of the model to explain previously published experimental data obtained using the PKA-based biosensor (12) and experimental data presented in the current manuscript, which were obtained using the Epac2-based biosensor. The size of these domains is consistent with previously published estimates of the size of submembrane compartments for cAMP made by Rich et al (23). The final domain is the bulk cytoplasmic compartment, which makes up the balance of the cytosolic volume and is associated with the remainder of the plasma membrane.…”

“…Other studies have used cyclic nucleotide-gated (CNG) ion channels as reporters of cAMP activity in the subsarcolemmal space of various cell types, including cardiac myocytes (23)(24)(25). Responses detected by isoforms of these channels that have an EC 50 for activation by cAMP of ϳ10 M suggest that levels can reach high micromolar concentrations (23).…”

Nucleotide-gated KATPchannels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

“…3A). This diffusion coefficient is in line with other estimates for diffusion of nucleotides in a cell, and indicates strong diffusion barriers between cellular compartments [34,41,42].…”

“…3A). This diffusion coefficient is in line with other estimates for diffusion of nucleotides in a cell, and indicates strong diffusion barriers between cellular compartments [34,41,42].The lower value of D obtained here in the presence of AK, compared to D = 2.3 10 -11 cm 2 /s reported previously in the absence of AK [34], underscores the significance of this enzyme in reducing ATP and ADP gradients between cell compartments. Actual nucleotide gradients at different bulk ATP levels can be obtained by solving Eqs 1, 2, 4 and 5: The nucleotide gradient for ATP is significantly lower in the presence (Eq.…”

The cyclic AMP phenotype of fragile X and autism

“…Cyclic AMP produces short term or long term changes in neuron function by acting respectively through PKA or an EPAC-Rap-MAPK cascade involving an exchange protein activated by cAMP (EPAC), a small GTPase (Rap1), and a mitogen-activated protein kinase (MAPK) (Neves, Ram et al 2002). Cyclic AMP subcellular distribution is not uniform (Rich, Fagan et al 2000) and its levels are tightly regulated by phosphodiesterases (PDEs). Regulation of cAMP levels is important because cAMP has several mechanisms to affect cellular function.…”