Cyclic AMP Diffusion Coefficient in Frog Olfactory Cilia

Abstract

Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli.

Distribution, Amplification, and Summation of Cyclic Nucleotide Sensitivities within Single Olfactory Sensory Cilia

“…The latter, voltage profile within the cilium, will be calculated and examined below. It has been reported for detached ciliary preparation that the cytoplasmic cAMP diffusion coefficient is 2.7 ϫ 10 Ϫ6 cm 2 /s (Chen et al, 1999). Therefore, with x (average movement) ϭ (2Dt) 1/2 , the total cilia (20 m length) could be filled with the cAMP within 1 s. However, experimental data observed here are inconsistent with such simple idea.…”

AKAP signaling complexes: getting to the heart of the matter

“…Although the diffusion of cAMP has long been considered to be fast within cells, recent studies have suggested that enzymes involved in cAMP synthesis and degradation generate boundaries for cAMP diffusion [7][8][9]16,17]. Studies in HEK293 cells where rat olfactory CNG channels, which have been modified to be sensitive to cAMP, were overexpressed have illustrated that distinct cAMP signals coexist within these cells [10].…”

Dynamics of Protein Kinase A Signaling at the Membrane, in the Cytosol, and in the Nucleus of Neurons in Mouse Brain Slices

“…cAMP diffusion from the membrane toward the cytosol can be one factor, but studies in frog olfactory cilia (Chen et al, 1999), cultured hippocampal neurons (Nikolaev et al, 2004), and cardiocytes (Saucerman et al, 2006) have shown a diffusion speed of tens of micrometers per second, which cannot account for the kinetic differences observed between the membrane compartment and the cytosol. Endogenous buffers may also contribute to slowing down the propagation of the signal, as suggested by modeling studies (Rich and Karpen, 2002;Saucerman et al, 2006).…”