Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Oct;83(4):1953–1964. doi: 10.1016/S0006-3495(02)73957-X

Pore topology of the hyperpolarization-activated cyclic nucleotide-gated channel from sea urchin sperm.

Paola Roncaglia 1, Pavel Mistrík 1, Vincent Torre 1
PMCID: PMC1302285  PMID: 12324414

Abstract

The current flow through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, referred to as I(h), plays a major role in several fundamental biological processes. The sequence of the presumed pore region of HCN channels is reminiscent of that of most known K(+)-selective channels. In the present work, the pore topology of an HCN channel from sea urchin sperm, called SpHCN, was investigated by means of the substituted-cysteine accessibility method (SCAM). The I(h) current in the wild-type (w.t.) SpHCN channel was irreversibly blocked by intracellular Cd(2+). This blockage was not observed in mutant C428S. Extracellular Cd(2+) did not cause any inhibition of the I(h) current in the w.t. SpHCN channel, but blocked the current in mutant channels K433C and F434C. Large extracellular anions blocked the current both in the w.t. and K433Q mutant channel. These results suggest that 1) cysteine in position 428 faces the intracellular medium; 2) lysine and phenylalanine in position 433 and 434, respectively, face the extracellular side of the membrane; and 3) lysine 433 does not mediate the anion blockade. Additionally, our study confirms that the K(+) channel signature sequence GYG also forms the inner pore in HCN channels.

Full Text

The Full Text of this article is available as a PDF (360.6 KB).

Selected References

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

  1. Becchetti A., Gamel K. The properties of cysteine mutants in the pore region of cyclic-nucleotide-gated channels. Pflugers Arch. 1999 Oct;438(5):587–596. doi: 10.1007/s004249900062. [DOI] [PubMed] [Google Scholar]
  2. Becchetti A., Gamel K., Torre V. Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines. J Gen Physiol. 1999 Sep;114(3):377–392. doi: 10.1085/jgp.114.3.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Becchetti A., Roncaglia P. Cyclic nucleotide-gated channels: intra- and extracellular accessibility to Cd2+ of substituted cysteine residues within the P-loop. Pflugers Arch. 2000 Aug;440(4):556–565. doi: 10.1007/s004240000324. [DOI] [PubMed] [Google Scholar]
  4. Capovilla M., Caretta A., Cervetto L., Torre V. Ionic movements through light-sensitive channels of toad rods. J Physiol. 1983 Oct;343:295–310. doi: 10.1113/jphysiol.1983.sp014893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chapman M. L., Krovetz H. S., VanDongen A. M. GYGD pore motifs in neighbouring potassium channel subunits interact to determine ion selectivity. J Physiol. 2001 Jan 1;530(Pt 1):21–33. doi: 10.1111/j.1469-7793.2001.0021m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clapham D. E. Not so funny anymore: pacing channels are cloned. Neuron. 1998 Jul;21(1):5–7. doi: 10.1016/s0896-6273(00)80508-5. [DOI] [PubMed] [Google Scholar]
  7. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol. 1993;55:455–472. doi: 10.1146/annurev.ph.55.030193.002323. [DOI] [PubMed] [Google Scholar]
  8. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  9. Eismann E., Müller F., Heinemann S. H., Kaupp U. B. A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1109–1113. doi: 10.1073/pnas.91.3.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fain G. L., Quandt F. N., Bastian B. L., Gerschenfeld H. M. Contribution of a caesium-sensitive conductance increase to the rod photoresponse. Nature. 1978 Mar 30;272(5652):466–469. doi: 10.1038/272467a0. [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. Flynn G. E., Zagotta W. N. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron. 2001 Jun;30(3):689–698. doi: 10.1016/s0896-6273(01)00324-5. [DOI] [PubMed] [Google Scholar]
  13. Frace A. M., Maruoka F., Noma A. Control of the hyperpolarization-activated cation current by external anions in rabbit sino-atrial node cells. J Physiol. 1992;453:307–318. doi: 10.1113/jphysiol.1992.sp019230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gauss R., Seifert R., Kaupp U. B. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature. 1998 Jun 11;393(6685):583–587. doi: 10.1038/31248. [DOI] [PubMed] [Google Scholar]
  15. Gauss R., Seifert R. Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels. Chronobiol Int. 2000 Jul;17(4):453–469. doi: 10.1081/cbi-100101057. [DOI] [PubMed] [Google Scholar]
  16. Gordon S. E., Zagotta W. N. A histidine residue associated with the gate of the cyclic nucleotide-activated channels in rod photoreceptors. Neuron. 1995 Jan;14(1):177–183. doi: 10.1016/0896-6273(95)90252-x. [DOI] [PubMed] [Google Scholar]
  17. Halliwell J. V., Adams P. R. Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Res. 1982 Oct 28;250(1):71–92. doi: 10.1016/0006-8993(82)90954-4. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Heginbotham L., Lu Z., Abramson T., MacKinnon R. Mutations in the K+ channel signature sequence. Biophys J. 1994 Apr;66(4):1061–1067. doi: 10.1016/S0006-3495(94)80887-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ho W. K., Brown H. F., Noble D. High selectivity of the i(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells. Pflugers Arch. 1994 Jan;426(1-2):68–74. doi: 10.1007/BF00374672. [DOI] [PubMed] [Google Scholar]
  21. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996 Feb;14(1):33-8, 27-8. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]
  22. Ishii T. M., Takano M., Xie L. H., Noma A., Ohmori H. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem. 1999 Apr 30;274(18):12835–12839. doi: 10.1074/jbc.274.18.12835. [DOI] [PubMed] [Google Scholar]
  23. Karlin A., Akabas M. H. Substituted-cysteine accessibility method. Methods Enzymol. 1998;293:123–145. doi: 10.1016/s0076-6879(98)93011-7. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Krieger J., Strobel J., Vogl A., Hanke W., Breer H. Identification of a cyclic nucleotide- and voltage-activated ion channel from insect antennae. Insect Biochem Mol Biol. 1999 Mar;29(3):255–267. doi: 10.1016/s0965-1748(98)00134-9. [DOI] [PubMed] [Google Scholar]
  26. Krovetz H. S., VanDongen H. M., VanDongen A. M. Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore. Biophys J. 1997 Jan;72(1):117–126. doi: 10.1016/S0006-3495(97)78651-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kubo Y., Yoshimichi M., Heinemann S. H. Probing pore topology and conformational changes of Kir2.1 potassium channels by cysteine scanning mutagenesis. FEBS Lett. 1998 Sep 11;435(1):69–73. doi: 10.1016/s0014-5793(98)01038-2. [DOI] [PubMed] [Google Scholar]
  28. Kürz L. L., Zühlke R. D., Zhang H. J., Joho R. H. Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys J. 1995 Mar;68(3):900–905. doi: 10.1016/S0006-3495(95)80266-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Laio A., Torre V. Physical origin of selectivity in ionic channels of biological membranes. Biophys J. 1999 Jan;76(1 Pt 1):129–148. doi: 10.1016/S0006-3495(99)77184-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ludwig A., Zong X., Jeglitsch M., Hofmann F., Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature. 1998 Jun 11;393(6685):587–591. doi: 10.1038/31255. [DOI] [PubMed] [Google Scholar]
  31. Ludwig A., Zong X., Stieber J., Hullin R., Hofmann F., Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J. 1999 May 4;18(9):2323–2329. doi: 10.1093/emboj/18.9.2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marbán E. Inaugural editorial: a new era for Circulation Research. Circ Res. 1999 Jul 9;85(1):1–3. doi: 10.1161/01.res.85.1.1. [DOI] [PubMed] [Google Scholar]
  33. Marx T., Gisselmann G., Störtkuhl K. F., Hovemann B. T., Hatt H. Molecular cloning of a putative voltage- and cyclic nucleotide-gated ion channel present in the antennae and eyes of Drosophila melanogaster. Invert Neurosci. 1999 2000;4(1):55–63. doi: 10.1007/pl00022368. [DOI] [PubMed] [Google Scholar]
  34. Miledi R., Parker I. Chloride current induced by injection of calcium into Xenopus oocytes. J Physiol. 1984 Dec;357:173–183. doi: 10.1113/jphysiol.1984.sp015495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nizzari M., Sesti F., Giraudo M. T., Virginio C., Cattaneo A., Torre V. Single-channel properties of cloned cGMP-activated channels from retinal rods. Proc Biol Sci. 1993 Oct 22;254(1339):69–74. doi: 10.1098/rspb.1993.0128. [DOI] [PubMed] [Google Scholar]
  36. Pape H. C. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol. 1996;58:299–327. doi: 10.1146/annurev.ph.58.030196.001503. [DOI] [PubMed] [Google Scholar]
  37. Pongs O., Kecskemethy N., Müller R., Krah-Jentgens I., Baumann A., Kiltz H. H., Canal I., Llamazares S., Ferrus A. Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 1988 Apr;7(4):1087–1096. doi: 10.1002/j.1460-2075.1988.tb02917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Root M. J., MacKinnon R. Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron. 1993 Sep;11(3):459–466. doi: 10.1016/0896-6273(93)90150-p. [DOI] [PubMed] [Google Scholar]
  39. Sali A., Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993 Dec 5;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
  40. Santoro B., Grant S. G., Bartsch D., Kandel E. R. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14815–14820. doi: 10.1073/pnas.94.26.14815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Santoro B., Liu D. T., Yao H., Bartsch D., Kandel E. R., Siegelbaum S. A., Tibbs G. R. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell. 1998 May 29;93(5):717–729. doi: 10.1016/s0092-8674(00)81434-8. [DOI] [PubMed] [Google Scholar]
  42. Santoro B., Tibbs G. R. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann N Y Acad Sci. 1999 Apr 30;868:741–764. doi: 10.1111/j.1749-6632.1999.tb11353.x. [DOI] [PubMed] [Google Scholar]
  43. Seifert R., Scholten A., Gauss R., Mincheva A., Lichter P., Kaupp U. B. Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9391–9396. doi: 10.1073/pnas.96.16.9391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sesti F., Eismann E., Kaupp U. B., Nizzari M., Torre V. The multi-ion nature of the cGMP-gated channel from vertebrate rods. J Physiol. 1995 Aug 15;487(1):17–36. doi: 10.1113/jphysiol.1995.sp020858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vaccari T., Moroni A., Rocchi M., Gorza L., Bianchi M. E., Beltrame M., DiFrancesco D. The human gene coding for HCN2, a pacemaker channel of the heart. Biochim Biophys Acta. 1999 Sep 3;1446(3):419–425. doi: 10.1016/s0167-4781(99)00092-5. [DOI] [PubMed] [Google Scholar]
  46. Wollmuth L. P., Hille B. Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders. J Gen Physiol. 1992 Nov;100(5):749–765. doi: 10.1085/jgp.100.5.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yellen G., Sodickson D., Chen T. Y., Jurman M. E. An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. Biophys J. 1994 Apr;66(4):1068–1075. doi: 10.1016/S0006-3495(94)80888-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhou Y., Morais-Cabral J. H., Kaufman A., MacKinnon R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature. 2001 Nov 1;414(6859):43–48. doi: 10.1038/35102009. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES