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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
. 1991 Nov 1;88(21):9868–9872. doi: 10.1073/pnas.88.21.9868

Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

W Altenhofen 1, J Ludwig 1, E Eismann 1, W Kraus 1, W Bönigk 1, U B Kaupp 1
PMCID: PMC52822  PMID: 1719541

Abstract

Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.

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Selected References

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

  1. Baker E. N., Hubbard R. E. Hydrogen bonding in globular proteins. Prog Biophys Mol Biol. 1984;44(2):97–179. doi: 10.1016/0079-6107(84)90007-5. [DOI] [PubMed] [Google Scholar]
  2. Breer H., Boekhoff I., Tareilus E. Rapid kinetics of second messenger formation in olfactory transduction. Nature. 1990 May 3;345(6270):65–68. doi: 10.1038/345065a0. [DOI] [PubMed] [Google Scholar]
  3. Chau V., Huang L. C., Romero G., Biltonen R. L., Huang C. Kinetic studies on the dissociation of adenosine cyclic 3',5'-monophosphate from the regulatory subunit of protein kinase from rabbit skeletal muscle. Biochemistry. 1980 Mar 4;19(5):924–928. doi: 10.1021/bi00546a016. [DOI] [PubMed] [Google Scholar]
  4. Cook N. J., Hanke W., Kaupp U. B. Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors. Proc Natl Acad Sci U S A. 1987 Jan;84(2):585–589. doi: 10.1073/pnas.84.2.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Delgado R., Hidalgo P., Diaz F., Latorre R., Labarca P. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):557–560. doi: 10.1073/pnas.88.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dhallan R. S., Yau K. W., Schrader K. A., Reed R. R. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature. 1990 Sep 13;347(6289):184–187. doi: 10.1038/347184a0. [DOI] [PubMed] [Google Scholar]
  7. DiFrancesco D., Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature. 1991 May 9;351(6322):145–147. doi: 10.1038/351145a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Garges S., Adhya S. Sites of allosteric shift in the structure of the cyclic AMP receptor protein. Cell. 1985 Jul;41(3):745–751. doi: 10.1016/s0092-8674(85)80055-6. [DOI] [PubMed] [Google Scholar]
  10. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  11. Haynes L., Yau K. W. Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones. Nature. 1985 Sep 5;317(6032):61–64. doi: 10.1038/317061a0. [DOI] [PubMed] [Google Scholar]
  12. Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. doi: 10.1093/nar/17.16.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaupp U. B., Niidome T., Tanabe T., Terada S., Bönigk W., Stühmer W., Cook N. J., Kangawa K., Matsuo H., Hirose T. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature. 1989 Dec 14;342(6251):762–766. doi: 10.1038/342762a0. [DOI] [PubMed] [Google Scholar]
  14. Kaupp U. B. The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium. Trends Neurosci. 1991 Apr;14(4):150–157. doi: 10.1016/0166-2236(91)90087-b. [DOI] [PubMed] [Google Scholar]
  15. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Landgraf W., Hofmann F. The amino terminus regulates binding to and activation of cGMP-dependent protein kinase. Eur J Biochem. 1989 May 15;181(3):643–650. doi: 10.1111/j.1432-1033.1989.tb14771.x. [DOI] [PubMed] [Google Scholar]
  18. Ludwig J., Margalit T., Eismann E., Lancet D., Kaupp U. B. Primary structure of cAMP-gated channel from bovine olfactory epithelium. FEBS Lett. 1990 Sep 17;270(1-2):24–29. doi: 10.1016/0014-5793(90)81226-e. [DOI] [PubMed] [Google Scholar]
  19. McKay D. B., Weber I. T., Steitz T. A. Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem. 1982 Aug 25;257(16):9518–9524. [PubMed] [Google Scholar]
  20. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Methfessel C., Witzemann V., Takahashi T., Mishina M., Numa S., Sakmann B. Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflugers Arch. 1986 Dec;407(6):577–588. doi: 10.1007/BF00582635. [DOI] [PubMed] [Google Scholar]
  22. Nakamura T., Gold G. H. A cyclic nucleotide-gated conductance in olfactory receptor cilia. 1987 Jan 29-Feb 4Nature. 325(6103):442–444. doi: 10.1038/325442a0. [DOI] [PubMed] [Google Scholar]
  23. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  24. Shabb J. B., Ng L., Corbin J. D. One amino acid change produces a high affinity cGMP-binding site in cAMP-dependent protein kinase. J Biol Chem. 1990 Sep 25;265(27):16031–16034. [PubMed] [Google Scholar]
  25. Shirley S. G., Robinson C. J., Dickinson K., Aujla R., Dodd G. H. Olfactory adenylate cyclase of the rat. Stimulation by odorants and inhibition by Ca2+. Biochem J. 1986 Dec 1;240(2):605–607. doi: 10.1042/bj2400605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stühmer W., Methfessel C., Sakmann B., Noda M., Numa S. Patch clamp characterization of sodium channels expressed from rat brain cDNA. Eur Biophys J. 1987;14(3):131–138. doi: 10.1007/BF00253837. [DOI] [PubMed] [Google Scholar]
  27. Tanaka J. C., Eccleston J. F., Furman R. E. Photoreceptor channel activation by nucleotide derivatives. Biochemistry. 1989 Apr 4;28(7):2776–2784. doi: 10.1021/bi00433a006. [DOI] [PubMed] [Google Scholar]
  28. Taylor S. S., Buechler J. A., Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem. 1990;59:971–1005. doi: 10.1146/annurev.bi.59.070190.004543. [DOI] [PubMed] [Google Scholar]
  29. Weber I. T., Shabb J. B., Corbin J. D. Predicted structures of the cGMP binding domains of the cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP and cGMP binding sites. Biochemistry. 1989 Jul 11;28(14):6122–6127. doi: 10.1021/bi00440a059. [DOI] [PubMed] [Google Scholar]
  30. Weber I. T., Steitz T. A., Bubis J., Taylor S. S. Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase. Biochemistry. 1987 Jan 27;26(2):343–351. doi: 10.1021/bi00376a003. [DOI] [PubMed] [Google Scholar]
  31. Yau K. W., Baylor D. A. Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu Rev Neurosci. 1989;12:289–327. doi: 10.1146/annurev.ne.12.030189.001445. [DOI] [PubMed] [Google Scholar]

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