European Biophysics Journal

, Volume 38, Issue 7, pp 993–1002

Conformational rearrangements in the S6 domain and C-linker during gating in CNGA1 channels

  • Anil V. Nair
  • Chuong H. H. Nguyen
  • Monica Mazzolini
Original Paper

Abstract

This work completes previous findings and, using cysteine scanning mutagenesis (CSM) and biochemical methods, provides detailed analysis of conformational changes of the S6 domain and C-linker during gating of CNGA1 channels. Specific residues between Phe375 and Val424 were mutated to a cysteine in the CNGA1 and CNGA1cys-free background and the effect of intracellular Cd2+ or cross-linkers of different length in the open and closed state was studied. In the closed state, Cd2+ ions inhibited mutant channels A406C and Q409C and the longer cross-linker reagent M-4-M inhibited mutant channels A406Ccys-free and Q409Ccys-free. Cd2+ ions inhibited mutant channels D413C and Y418C in the open state, both constructed in a CNGA1 and CNGA1cys-free background. Our results suggest that, in the closed state, residues from Phe375 to approximately Ala406 form a helical bundle with a three-dimensional (3D) structure similar to those of the KcsA; furthermore, in the open state, residues from Ser399 to Gln409 in homologous subunits move far apart, as expected from the gating in K+ channels; in contrast, residues from Asp413 to Tyr418 in homologous subunits become closer in the open state.

Keywords

Gating CNGA1 channels Cd2+ inhibition Pore S6 domain, C-linker 

Abbreviations

CNG

Cyclic nucleotide-gated

CNBD

Cyclic nucleotide-binding domain

CSM

Cysteine scanning mutagenesis

MTS

Methanethiosulfonate

MTSET

2-(Trimethylammonium)ethyl] methanethiosulfonate bromide

MTSPT

3-(Trimethylammonium)propyl methanethiosulfonate bromide

MTS-PtrEA

3-(Triethylammonium)propyl methanthiosulfonate bromide

M-2-M

1,2-Ethanediyl bismethanethiosulfonate

M-4-M

1,4-Butanediyl bismethanethiosulfonate

M-6-M

1,6-Hexanediyl bismethanethiosulfonate

Supplementary material

249_2009_491_MOESM1_ESM.doc (88 kb)
Supplementary material 1 (DOC 88 kb)

References

  1. Akabas MH, Stauffer DA, Xu M, Karlin A (1992) Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258:307–310. doi:10.1126/science.1384130 PubMedCrossRefGoogle Scholar
  2. Becchetti A, Roncaglia P (2000) Cyclic nucleotide-gated channels: intra- and extracellular accessibility to Cd2+ of substituted cysteine residues within the P-loop. Pflugers Arch 440:556–565PubMedGoogle Scholar
  3. Becchetti A, Gamel K, Torre V (1999) Cyclic nucleotide-gated channels: pore topology studied through the accessibility of reporter cysteines. J Gen Physiol 114:377–392. doi:10.1085/jgp.114.3.377 PubMedCrossRefGoogle Scholar
  4. Benitah JP, Tomaselli GF, Marban E (1996) Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis. Proc Natl Acad Sci USA 93:7392–7396. doi:10.1073/pnas.93.14.7392 PubMedCrossRefGoogle Scholar
  5. Biel M, Zong X, Ludwig A, Sautter A, Hofmann F (1999) Structure and function of cyclic nucleotide-gated channels. Rev Physiol Biochem Pharmacol 135:151–171. doi:10.1007/BFb0033672 PubMedCrossRefGoogle Scholar
  6. Bradley J, Frings S, Yau KW, Reed R (2001) Nomenclature for ion channel subunits. Science 294:2095–2096. doi:10.1126/science.294.5549.2095 PubMedCrossRefGoogle Scholar
  7. Brown RL, Snow SD, Haley TL (1998) Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels. Biophys J 75:825–833. doi:10.1016/S0006-3495(98)74064-0 PubMedCrossRefGoogle Scholar
  8. Bucossi G, Eismann E, Sesti F, Nizzari M, Seri M, Kaupp UB, Torre V (1996) Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes. J Physiol 493(Pt 2):409–418PubMedGoogle Scholar
  9. Chen TY, Illing M, Molday LL, Hsu YT, Yau KW, Molday RS (1994) Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca2+-calmodulin modulation. Proc Natl Acad Sci USA 91:11757–11761. doi:10.1073/pnas.91.24.11757 PubMedCrossRefGoogle Scholar
  10. Craven KB, Zagotta WN (2006) CNG and HCN channels: Two Peas, One Pod. Annu Rev Physiol 68:375–401. doi:10.1146/annurev.physiol.68.040104.134728 PubMedCrossRefGoogle Scholar
  11. Craven KB, Olivier NB, Zagotta WN (2008) C-terminal movement during gating in cyclicc nucleotide-modulated channels. J Biol Chem 283(21):14728–14738. doi:10.1074/jbc.M710463200 PubMedCrossRefGoogle Scholar
  12. Doyle DA, Morais CJ, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77. doi:10.1126/science.280.5360.69 PubMedCrossRefGoogle Scholar
  13. Fesenko EE, Kolesnikov SS, Lyubarsky AL (1985) Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313:310–313. doi:10.1038/313310a0 PubMedCrossRefGoogle Scholar
  14. Flynn GE, Zagotta WN (2001) Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron 30:689–698. doi:10.1016/S0896-6273(01)00324-5 PubMedCrossRefGoogle Scholar
  15. Flynn GE, Zagotta WN (2003) A cysteine scan of the inner vestibule of cyclic nucleotide-gated channels reveals architecture and rearrangement of the pore. J Gen Physiol 121:563–582. doi:10.1085/jgp.200308819 PubMedCrossRefGoogle Scholar
  16. Giorgetti A, Nair AV, Codega P, Torre V, Carloni P (2005) Structural basis of gating of CNG channels. FEBS Lett 579:1968–1972. doi:10.1016/j.febslet.2005.01.086 PubMedCrossRefGoogle Scholar
  17. Hua L, Gordon SE (2005) Functional interactions between A’ helices in the C-linker of open CNG channels. J Gen Physiol 125:335–344. doi:10.1085/jgp.200409187 PubMedCrossRefGoogle Scholar
  18. Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R (2002a) Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417:515–522. doi:10.1038/417515a PubMedCrossRefGoogle Scholar
  19. Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R (2002b) The open pore conformation of potassium channels. Nature 417:523–526. doi:10.1038/417523a PubMedCrossRefGoogle Scholar
  20. Johnson JP, Zagotta WN (2001) Rotational movement during cyclic nucleotide-gated channel opening. Nature 412:917–921. doi:10.1038/35091089 PubMedCrossRefGoogle Scholar
  21. Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145. doi:10.1016/S0076-6879(98)93011-7 PubMedCrossRefGoogle Scholar
  22. Kaupp UB, Seifert R (2002) Cyclic nucleotide-gated ion channels. Physiol Rev 82:769–824PubMedGoogle Scholar
  23. Kaupp UB, Niidome T, Tanabe T, Terada S, Bonigk W, Stuhmer W, Cook NJ, Kangawa K, Matsuo H, Hirose T (1989) Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342:762–766. doi:10.1038/342762a0 PubMedCrossRefGoogle Scholar
  24. Körschen HG, Illing M, Seifert R, Sesti F, Williams A, Gotzes S, Colville C, Müller F, Dose A, Godde M (1995) A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor. Neuron 15:627–636. doi:10.1016/0896-6273(95)90151-5 PubMedCrossRefGoogle Scholar
  25. Krovetz HS, VanDongen HM, VanDongen AM (1997) Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore. Biophys J 72:117–126. doi:10.1016/S0006-3495(97)78651-X PubMedCrossRefGoogle Scholar
  26. Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, Cuthbertson J, Ashcroft FM, Ezaki T, Doyle DA (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300:1922–1926. doi:10.1126/science.1085028 PubMedCrossRefGoogle Scholar
  27. Kurz LL, Zuhlke RD, Zhang HJ, Joho RH (1995) Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys J 68:900–905. doi:10.1016/S0006-3495(95)80266-3 PubMedCrossRefGoogle Scholar
  28. Long SB, Campbell EB, MacKinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903. doi:10.1126/science.1116269 PubMedCrossRefGoogle Scholar
  29. Loo TW, Clarke DM (2001) Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers. J Biol Chem 276:36877–36880. doi:10.1074/jbc.C100467200 PubMedCrossRefGoogle Scholar
  30. Matulef K, Flynn GE, Zagotta WN (1999) Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron 24:443–452. doi:10.1016/S0896-6273(00)80857-0 PubMedCrossRefGoogle Scholar
  31. Mazzolini M, Punta M, Torre V (2002) Movement of the C-helix during the gating of cyclic nucleotide-gated channels. Biophys J 83:3283–3295. doi:10.1016/S0006-3495(02)75329-0 PubMedCrossRefGoogle Scholar
  32. Mazzolini M, Nair AV, Torre V (2008) A comparison of electrophysiological properties of the CNGA1, CNGA1tandem and CNGA1cys-free channels. Eur Biophys J 37(6):947–959. doi:10.1007/s00249-008-0312-1 PubMedCrossRefGoogle Scholar
  33. Mazzolini M, Anselmi C, Torre V (2009) The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating. J Gen Physiol 133(4):375–386. doi:10.1085/jgp.200810157 PubMedCrossRefGoogle Scholar
  34. Nair AV, Mazzolini M, Codega P, Giorgetti A, Torre V (2006) Locking CNGA1 channels in the open and closed state. Biophys J 90:3599–3607. doi:10.1529/biophysj.105.073346 PubMedCrossRefGoogle Scholar
  35. Nair AV, Anselmi C, Mazzolini M (2009) Movements of native C505 during channel gating in CNGA1 channels. Eur Biophys J 38(4):465–478. doi:10.1007/s00249-008-0396-7 PubMedCrossRefGoogle Scholar
  36. Nakamura T, Gold GH (1987) A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325:442–444. doi:10.1038/325442a0 PubMedCrossRefGoogle Scholar
  37. Roncaglia P, Becchetti A (2001) Cyclic-nucleotide-gated channels: pore topology in desensitizing E19A mutants. Pflugers Arch 441:772–780. doi:10.1007/s004240000480 PubMedCrossRefGoogle Scholar
  38. Rosenbaum T, Gordon SE (2002) Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels. Neuron 33:703–713. doi:10.1016/S0896-6273(02)00599-8 PubMedCrossRefGoogle Scholar
  39. Rothberg BS, Shin KS, Phale PS, Yellen G (2002) Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. J Gen Physiol 119:83–91. doi:10.1085/jgp.119.1.83 PubMedCrossRefGoogle Scholar
  40. Rothberg BS, Shin KS, Yellen G (2003) Movements near the gate of a hyperpolarization-activated cation channel. J Gen Physiol 122:501–510. doi:10.1085/jgp.200308928 PubMedCrossRefGoogle Scholar
  41. Shammat IM, Gordon SE (1999) Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels. Neuron 23:809–819. doi:10.1016/S0896-6273(01)80038-6 PubMedCrossRefGoogle Scholar
  42. Zagotta WN, Siegelbaum SA (1996) Structure and function of cyclic nucleotide-gated channels. Annu Rev Neurosci 19:235–263. doi:10.1146/annurev.ne.19.030196.001315 PubMedCrossRefGoogle Scholar
  43. Zheng J, Trudeau MC, Zagotta WN (2002) Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron 36:891–896. doi:10.1016/S0896-6273(02)01099-1 PubMedCrossRefGoogle Scholar
  44. Zhong H, Molday LL, Molday RS, Yau KW (2002) The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry. Nature 420:193–198. doi:10.1038/nature01201 PubMedCrossRefGoogle Scholar
  45. Zimmerman AL, Yamanaka G, Eckstein F, Baylor DA, Stryer L (1985) Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments. Proc Natl Acad Sci USA 82:8813–8817. doi:10.1073/pnas.82.24.8813 PubMedCrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Anil V. Nair
    • 1
    • 2
  • Chuong H. H. Nguyen
    • 1
  • Monica Mazzolini
    • 1
    • 3
  1. 1.International School for Advanced StudiesTriesteItaly
  2. 2.Department of PhysiologyNijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical CentreNijmegenThe Netherlands
  3. 3.International School for Advanced StudiesTriesteItaly

Personalised recommendations