Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes

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Abstract

Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5% of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607.). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, D(x), of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, D(x), of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8- (fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was ~6 min. Fluorescence images of rods were analyzed with a one- dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of D(x) of 1.9 and 1.0 μm2 · s-1 for the two dyes. It is shown that these results place an upper limit on D(x) for cGMP of 11 μm2 · s-1. The actual value of D(x) for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, D(x) falls within the range of 1.4-5.5 μm2 · s-1.

Original languageEnglish (US)
Pages (from-to)1335-1352
Number of pages18
JournalBiophysical Journal
Volume65
Issue number3
StatePublished - 1993
Externally publishedYes

Fingerprint

Rod Cell Outer Segment
Urodela
Cyclic GMP
Fluorescent Dyes
Electrodes
Coloring Agents
Amphibians
Primates
Fluorescence

ASJC Scopus subject areas

  • Biophysics

Cite this

Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes. / Olson, A.; Pugh Jr, Edward N.

In: Biophysical Journal, Vol. 65, No. 3, 1993, p. 1335-1352.

Research output: Contribution to journalArticle

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abstract = "Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5{\%} of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607.). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, D(x), of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, D(x), of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8- (fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was ~6 min. Fluorescence images of rods were analyzed with a one- dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of D(x) of 1.9 and 1.0 μm2 · s-1 for the two dyes. It is shown that these results place an upper limit on D(x) for cGMP of 11 μm2 · s-1. The actual value of D(x) for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, D(x) falls within the range of 1.4-5.5 μm2 · s-1.",
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AB - Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5% of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607.). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, D(x), of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, D(x), of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8- (fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was ~6 min. Fluorescence images of rods were analyzed with a one- dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of D(x) of 1.9 and 1.0 μm2 · s-1 for the two dyes. It is shown that these results place an upper limit on D(x) for cGMP of 11 μm2 · s-1. The actual value of D(x) for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, D(x) falls within the range of 1.4-5.5 μm2 · s-1.

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