Cyclic GMP was introduced into isolated salamander rods through a tight-seal electrode attached to the inner segment while the outer segment was held in a suction electrode; nucleotide-induced membrane current was recorded by both electrodes. After 3-15 s of nucleotide exposure the cells were stimulated with intense, brief flashes, which suppressed 90-95% of the induced membrane current. The magnitude of the induced light-sensitive current depended little on the pipette cyclic GMP concentration in the range 10-20 mM: the mean whole-cell current magnitude was 1256 ± 160 pA (mean ± 2 S.E.M., n = 41). Experiments and analyses addressed hypotheses about the nature of the magnitude limitation on the induced current. It was shown that the spatial distribution of nucleotide, the residual series resistance of the whole-cell electrode, and the diminution of the ion gradients driving the induced current did not limit the current magnitude by more than 20%. In contrast, the hypothesis that outer segment internal longitudinal resistance severely limits the magnitude of the cyclic GMP-induced current was supported by experiments in which various lengths of the outer segment were drawn into the suction electrode. These showed that the ratio of nucleotide-induced light-sensitive current collected by the suction electrode to that collected by the whole-cell electrode decreased steeply as a function of outer segment length excluded from the suction electrode, having an apparent space constant of 5-7 μm. A cable model of the rod was developed and used to analyse the magnitude of the nucleotide-induced currents. The data are consistent with an outer segment longitudinal resistance of 1-4 MΩ μm-1, and a maximum (space-clamped) light-sensitive current density of 323 pA μm-1, equivalent to a total induced current of 7200 pA (23 μm outer segment). A diffusion model was developed and combined with the non-linear cable model to provide an account for the time course of the induced membrane currents. The results are consistent with an effective longitudinal diffusion coefficient of cyclic GMP in the outer segment of 3-10 μm2 s-1, and Hill coefficient of 2-3 for the cyclic GMP gating of the light-sensitive conductance. 8-Bromo-cyclic GMP also caused the light-sensitive membrane current to increase to about the same magnitude as did cyclic GMP. However, 1/5 to 1/10 as much 8-bromo-cyclic GMP in the tight-seal pipette caused about the same rate of increase of light-sensitive membrane current as a given amount of cyclic GMP, and the induced current was far less sensitive to light.
|Original language||English (US)|
|Number of pages||21|
|Journal||Journal of Physiology|
|State||Published - 1990|
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