Diffusion of fluorescent cyclic GMP analogs introduced from a whole-cell electrode in rod outer segments of the tiger salamander Ambystoma tigrinum

In rod photoreceptors, the degree of longitudinal spread of activation is critically dependent on the rate of diffusion of cyclic GMP, the internal messenger of phototransduction. The outer segment is filled with a stack of lamellar disks, which contain the proteins involved in the phototransduction cascade. The disks occupy more than 98% of the cross-sectional area of the outer segment, and thus present a barrier to longitudinal diffusion. The phosphodiesterase which hydrolyzes cyclic GMP is attached to the lamellar disks. The activity of phosphodiesterase creates a local sink to which cyclic GMP diffuses. This diffusion step, in part, determines the spatial extent of the photoresponse. Previous measurements of the apparent longitudinal diffusion coefficient (D$\sb{x}$) for cyclic GMP range over an order to magnitude, from 3-30 $\mu$m$\sp2\cdot\rm s\sp{-1}$. I have measured D$\sb{x}$ for two fluorescent cyclic GMP analogs: 5/6-carboxyfluorescein (CF) and 8(fluoresceinyl)thioguanosine 3$\sp\prime$5$\sp\prime$-cyclic monophosphate (fl-cGMP), introduced into detached outer segments via a whole-cell electrode. The spatial profile of fluorescence intensity over time was monitored and quantified with a digital image analysis system A model based on our solution of the one-dimensional diffusion equation, under the appropriate initial and boundary conditions, was used to estimate D$\sb{x}$ from the measured fluorescence intensity profiles. The mean D$\sb{x}$ ($\pm$ S.E.M.) for CF was 1.9 $\pm$ 0.4 $\mu\rm m\sp2\cdot\rm s\sp{-1}$ (N = 7) and for fl-cGMP, 0.95 $\pm$ 0.22 $\mu\rm m\sp2\cdot\rm s\sp{-1}$ (N = 9). Our measurements suggest that D$\sb{x}$ for cyclic GMP lies between 1.2-10.6 $\mu\rm m\sp2\cdot\rm s\sp{-1}$. Our results also suggest that there may be low-affinity binding sites for cGMP in addition to the known binding sites on the rod phosphodiesterase