Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Dec;82(24):8813–8817. doi: 10.1073/pnas.82.24.8813

Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.

A L Zimmerman, G Yamanaka, F Eckstein, D A Baylor, L Stryer
PMCID: PMC391528  PMID: 2417228

Abstract

cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.

Full text

PDF
8813

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baylor D. A., Lamb T. D., Yau K. W. Responses of retinal rods to single photons. J Physiol. 1979 Mar;288:613–634. [PMC free article] [PubMed] [Google Scholar]
  2. Baylor D. A., Lamb T. D., Yau K. W. The membrane current of single rod outer segments. J Physiol. 1979 Mar;288:589–611. [PMC free article] [PubMed] [Google Scholar]
  3. Capovilla M., Cervetto L., Torre V. The effect of phosphodiesterase inhibitors on the electrical activity of toad rods. J Physiol. 1983 Oct;343:277–294. doi: 10.1113/jphysiol.1983.sp014892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caretta A., Cavaggioni A. Fast ionic flux activated by cyclic GMP in the membrane of cattle rod outer segments. Eur J Biochem. 1983 Apr 15;132(1):1–8. doi: 10.1111/j.1432-1033.1983.tb07317.x. [DOI] [PubMed] [Google Scholar]
  5. Caretta A. Effect of cGMP and cations on the permeability of cattle retinal disks. Eur J Biochem. 1985 May 2;148(3):599–606. doi: 10.1111/j.1432-1033.1985.tb08882.x. [DOI] [PubMed] [Google Scholar]
  6. Chabre M. Trigger and amplification mechanisms in visual phototransduction. Annu Rev Biophys Biophys Chem. 1985;14:331–360. doi: 10.1146/annurev.bb.14.060185.001555. [DOI] [PubMed] [Google Scholar]
  7. Cobbs W. H., Pugh E. N., Jr Cyclic GMP can increase rod outer-segment light-sensitive current 10-fold without delay of excitation. Nature. 1985 Feb 14;313(6003):585–587. doi: 10.1038/313585a0. [DOI] [PubMed] [Google Scholar]
  8. Cote R. H., Biernbaum M. S., Nicol G. D., Bownds M. D. Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors. J Biol Chem. 1984 Aug 10;259(15):9635–9641. [PubMed] [Google Scholar]
  9. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  10. Fazakerley G. V., Russell J. C., Wolfe M. A. Determination of the syn-anti equilibrium of some purine 3':5'-nucleotides by nuclear-magnetic-relaxation perturbation in the presence of a lanthanide-ion probe. Eur J Biochem. 1977 Jun 15;76(2):601–605. doi: 10.1111/j.1432-1033.1977.tb11630.x. [DOI] [PubMed] [Google Scholar]
  11. Fesenko E. E., Kolesnikov S. S., Lyubarsky A. L. Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature. 1985 Jan 24;313(6000):310–313. doi: 10.1038/313310a0. [DOI] [PubMed] [Google Scholar]
  12. Fung B. K., Hurley J. B., Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A. 1981 Jan;78(1):152–156. doi: 10.1073/pnas.78.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagins W. A., Penn R. D., Yoshikami S. Dark current and photocurrent in retinal rods. Biophys J. 1970 May;10(5):380–412. doi: 10.1016/S0006-3495(70)86308-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurley J. B., Stryer L. Purification and characterization of the gamma regulatory subunit of the cyclic GMP phosphodiesterase from retinal rod outer segments. J Biol Chem. 1982 Sep 25;257(18):11094–11099. [PubMed] [Google Scholar]
  15. Koch K. W., Kaupp U. B. Cyclic GMP directly regulates a cation conductance in membranes of bovine rods by a cooperative mechanism. J Biol Chem. 1985 Jun 10;260(11):6788–6800. [PubMed] [Google Scholar]
  16. MacLeish P. R., Schwartz E. A., Tachibana M. Control of the generator current in solitary rods of the Ambystoma tigrinum retina. J Physiol. 1984 Mar;348:645–664. doi: 10.1113/jphysiol.1984.sp015131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matthews H. R., Torre V., Lamb T. D. Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods. Nature. 1985 Feb 14;313(6003):582–585. doi: 10.1038/313582a0. [DOI] [PubMed] [Google Scholar]
  18. Miki N., Baraban J. M., Keirns J. J., Boyce J. J., Bitensky M. W. Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. J Biol Chem. 1975 Aug 25;250(16):6320–6327. [PubMed] [Google Scholar]
  19. Miller J. P., Boswell K. H., Muneyama K., Simon L. N., Robins R. K., Shuman D. A. Synthesis and biochemical studies of various 8-substituted derivatives of guanosine 3',5'-cyclic phosphate, inosine 3',5'-cyclic phosphate, and xanthosine 3',5'-cyclic phosphate. Biochemistry. 1973 Dec 18;12(26):5310–5319. doi: 10.1021/bi00750a014. [DOI] [PubMed] [Google Scholar]
  20. Senter P. D., Eckstein F., Mülsch A., Böhme E. The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase. J Biol Chem. 1983 Jun 10;258(11):6741–6745. [PubMed] [Google Scholar]
  21. Tavale S. S., Sobell H. M. Crystal and molecular structure of 8-bromoguanosine and 8-bromoadenosine, two purine nucleosides in the syn conformation. J Mol Biol. 1970 Feb 28;48(1):109–123. doi: 10.1016/0022-2836(70)90222-6. [DOI] [PubMed] [Google Scholar]
  22. Werblin F. S. Transmission along and between rods in the tiger salamander retina. J Physiol. 1978 Jul;280:449–470. doi: 10.1113/jphysiol.1978.sp012394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yau K. W., McNaughton P. A., Hodgkin A. L. Effect of ions on the light-sensitive current in retinal rods. Nature. 1981 Aug 6;292(5823):502–505. doi: 10.1038/292502a0. [DOI] [PubMed] [Google Scholar]
  24. Yau K. W., Nakatani K. Electrogenic Na-Ca exchange in retinal rod outer segment. Nature. 1984 Oct 18;311(5987):661–663. doi: 10.1038/311661a0. [DOI] [PubMed] [Google Scholar]
  25. Yee R., Liebman P. A. Light-activated phosphodiesterase of the rod outer segment. Kinetics and parameters of activation and deactivation. J Biol Chem. 1978 Dec 25;253(24):8902–8909. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES