Advertisement

Archives of Microbiology

, Volume 192, Issue 7, pp 595–602 | Cite as

A high-conductance cation channel from the inner membrane of the free-living soil bacteria Rhizobium etli

  • Daniel BallezaEmail author
  • Carmen Quinto
  • David Elias
  • Froylán Gómez-LagunasEmail author
Short Communication
  • 106 Downloads

Abstract

In this communication we reported the study of a cation channel present in the cytoplasmic membrane of the nitrogen fixing bacterium Rhizobium etli. Inner-membrane (IM) vesicles were purified and fused into planar lipid bilayers (PLBs), under voltage clamp conditions. We have found that fusion of IM-enriched vesicle fractions with these model membranes leads, mainly (>30% of 46 experiments), to the reconstitution of high-conductance channels. Following this strategy, the activity of a channel with main open conductance of 198 pS, in symmetrical 100 mM KCl, was recorded. The single-channel conductance increase to 653 pS in the presence of a 5:1 (cis to trans) gradient of KCl. The channel exhibits voltage dependency and a weak selectivity for cations showing a permeability ratios of P Rb/P K = 0.96, P Na/P K = 0.07, and a conductance ratio of γRbK = 1.1. The channel here characterized represents a previously undescribed Rhizobium channel although its precise role in rhizobial physiology remains yet to be determined.

Keywords

Rhizobium Ion channels Permeation 

Notes

Acknowledgments

We thank Guadalupe Zavala for her excellent technical assistance with electron microscopy and to Karla García y García for helping us with database searching using RetliDB. This work was supported by Grants CONACyT 56631 and DGAPA IN204907 to C.Q. and CONACyT 60313 and DGAPA IN222208 to F.G.L. D. Balleza thanks to DGAPA-UNAM for a postdoctoral fellowship. We sincerely thank the reviewers for their insightful comments and suggestions.

Supplementary material

203_2010_587_MOESM1_ESM.tif (985 kb)
Fig. S1. Sequence alignment of the putative transmembrane domains of the two proteins with similarity to K+ channels from R. etli (ABC91945 and YP467785), the cyclic nucleotide-regulated channel MlotiK1 (M. loti, Q98GN8), KvAp (Aeropyrum pernix, BAA79939), KcsA (Streptomyces lividans, CAA86025), Kv channel Shaker (Drosophila melanogaster, P08510), and RatKv2.1 (Rattus norvegicus, P15387). The secondary structure of Shaker is indicated. In sequences of prokaryotic and eukaryotic Kv channels, regions of high homology are coloured in grey, positively charged amino acids in S4 are in yellow whereas identical residues are in black boxes. Alignment was made with ClustalW followed by manual adjustment and exclusion of loops (TIFF 985 kb)

References

  1. Andersen C, Hughes C, Koronakis V (2002) Electrophysiological behavior of the TolC channel-tunnel in planar lipid bilayers. J Membr Biol 185:83–92CrossRefPubMedGoogle Scholar
  2. Balleza D, Gómez-Lagunas F, Quinto C (2010) Cloning and functional expression of an MscL ortholog from Rhizobium etli: characterization of a mechanosensitive channel. J Membr Biol 234:13–27CrossRefPubMedGoogle Scholar
  3. Barabote RD, Tamang DG, Abeywardena SN, Fallah NS, Fu JY, Lio JK, Mirhosseini P, Pezeshk R, Podell S, Salampessy ML, Thever MD, Saier MH Jr (2006) Extra domains in secondary transport carriers and channel proteins. Biochim Biophys Acta 1758:1557–1579CrossRefPubMedGoogle Scholar
  4. Baslè A, Iyer R, Delcour AH (2004) Subconductance states in OmpF gating. Biochim Biophys Acta 1664:100–107CrossRefPubMedGoogle Scholar
  5. Berrier C, Coulombe A, Houssin C, Ghazi A (1993) Voltage-dependent cationic channel of Escherichia coli. J Membr Biol 133:119–127PubMedGoogle Scholar
  6. Bezanilla F (1985) A high capacity data recording device based on a digital audio processor and a video cassette recorder. Biophys J 47:437–441CrossRefPubMedGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Cheng WWL, Enkvetchakul D, Nichols CG (2009) KirBac1.1: It’s an inward rectifying potassium channel. J Gen Physiol 133:295–305CrossRefPubMedGoogle Scholar
  9. Chiu PL, Pagel MD, Evans J, Chou HT, Zeng X, Gipson B, Stahlberg H, Nimigean CM (2007) The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MlotiK1 at 16 Ǻ resolution. Structure 15:1053–1064CrossRefPubMedGoogle Scholar
  10. Clayton GM, Altieri S, Heginbotham L, Unger VM, Morais-Cabral JH (2008) Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel. Proc Natl Acad Sci USA 105:1511–1515CrossRefPubMedGoogle Scholar
  11. Cordero-Morales JF, Cuello LG, Perozo E (2006) Voltage-dependent gating at the KcsA selectivity filter. Nat Struct Mol Biol 13:319–322CrossRefPubMedGoogle Scholar
  12. Cukierman S, Yellen G, Miller C (1985) The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block. Biophys J 48:477–484CrossRefPubMedGoogle Scholar
  13. de Maagd R, van Rossum C, Lugtenberg BJ (1988) Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides. J Bacteriol 170:3782–3785PubMedGoogle Scholar
  14. Eisenman G, Latorre R, Miller C (1986) Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle. Biophys J 50:1025–1034CrossRefPubMedGoogle Scholar
  15. Gibson KE, Kobayashi H, Walker GC (2008) Molecular determinants of a symbiotic chronic infection. Annu Rev Genet 42:413–441CrossRefPubMedGoogle Scholar
  16. Gonzalez V, Santamaria RI, Bustos P, Hernandez-Gonzalez I, Medrano-Soto A, Moreno-Hagelsieb G, Janga SC, Ramirez MA, Jimenez-Jacinto V, Collado-Vides J, Davila G (2006) The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc Natl Acad Sci USA 103:3834–3839CrossRefPubMedGoogle Scholar
  17. Hamill OP, McBride DW (1996) The pharmacology of mechanogated membrane ion channels. Pharmacol Rev 48:231–252PubMedGoogle Scholar
  18. Hayat MA (2000) Principles and techniques of electron microscopy. Biological applications, 4th edn. Van Nostrand Reinhold Co, New YorkGoogle Scholar
  19. Heginbotham L, MacKinnon R (1993) Conduction properties of the cloned Shaker K+ channel. Biophys J 65:2089–2096CrossRefPubMedGoogle Scholar
  20. Heginbotham L, Lu Z, Abramson T, MacKinnon R (1994) Mutations in the K+ channel signature sequence. Biophys J 66:1061–1067CrossRefPubMedGoogle Scholar
  21. Hernandez-Lucas I, Ramirez-Trujillo JA, Gaitan MA, Gu X, Flores M, Martinez-Romero E, Perez-Rueda E, Mavingui P (2006) Isolation and characterization of functional insertion sequences of rhizobia. FEMS Microbiol Lett 261:25–31CrossRefPubMedGoogle Scholar
  22. Hille B (2001) Ionic channels of excitable membranes, 3rd edn. Sinauer Associates, SunderlandGoogle Scholar
  23. Hu SL, Yamamoto Y, Kao CY (1989) Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte. J Gen Physiol 94:849–862CrossRefPubMedGoogle Scholar
  24. Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, MacKinnon R (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423:33–41CrossRefPubMedGoogle Scholar
  25. Knowles BH, Blatt MR, Tester M, Hornsell JM, Carroll J, Menestrina G, Ellar DJ (1989) A cytolytic δ-endotoxin from Bacillus thuringensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Lett 244:259–262CrossRefPubMedGoogle Scholar
  26. Kuo MM, Haynes WJ, Loukin SH, Kung C, Saimi Y (2005) Prokaryotic K(+) channels: from crystal structures to diversity. FEMS Microbiol Rev 29:961–985CrossRefPubMedGoogle Scholar
  27. Lewis BD, Spalding EP (1998) Nonselective block by La3+ of Arabidopsis ion channels involved in signal transduction. J Membr Biol 162:81–90CrossRefPubMedGoogle Scholar
  28. Martinac B, Saimi Y, Kung C (2008) Ion channels in microbes. Physiol Rev 88:1449–1490CrossRefPubMedGoogle Scholar
  29. Moris M, Braeken K, Schoeters E, Verreth C, Beullens S, Vanderleyden J, Michiels J (2005) Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp. J Bacteriol 187:5460–5469CrossRefPubMedGoogle Scholar
  30. Müeller P, Rudin DO (1969) Bimolecular lipid membranes: techniques of formation, study of electrical properties, and induction of gating phenomena. In: Passow M, Stampfli R (eds) Laboratory techniques of membrane biophysics. Springer, BerlinGoogle Scholar
  31. Negoda A, Xian M, Reusch RN (2007) Insight into the selectivity and gating functions of Streptomyces lividans KcsA. Proc Natl Acad Sci USA 104:4342–4346CrossRefPubMedGoogle Scholar
  32. Nimigean CM, Shane T, Miller C (2004) A cyclic nucleotide modulated prokaryotic K+ channel. J Gen Physiol 124:203–210CrossRefPubMedGoogle Scholar
  33. Noskov SY, Im W, Roux B (2004) Ion permeation through the μ-hemolysin channel: theoretical studies based on Brownian dynamics and Poisson-Nernst-Plank electrodiffusion theory. Biophys J 87:2299–2309CrossRefPubMedGoogle Scholar
  34. Parker MW, Feil SC (2005) Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88:91–142CrossRefPubMedGoogle Scholar
  35. Paulsen IT, Nguyen L, Sliwinski MK, Rabus R, Saier MH Jr (2000) Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes. J Mol Biol 301:75–100CrossRefPubMedGoogle Scholar
  36. Rodríguez-Navarro A (2000) Potassium transport in fungi and plants. Biochim Biophys Acta 1469:1–30PubMedGoogle Scholar
  37. Ropele M, Menestrina G (1989) Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes. Biochim Biophys Acta 985:9–18CrossRefPubMedGoogle Scholar
  38. Ruta V, Jiang Y, Lee A, Chen J, MacKinnon R (2003) Functional analysis of an archaebacterial voltage-dependent K+ channel. Nature 422:180–185CrossRefPubMedGoogle Scholar
  39. Schnaitman CA (1970) Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol 104:890–901PubMedGoogle Scholar
  40. Siemen D, Loupatatzis C, Borecky J, Gulbins E, Lang F (1999) Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line. Biochem Biophys Res Comm 257:549–554CrossRefPubMedGoogle Scholar
  41. Silverman WR, Heginbotham L (2007) The MlotiK1 channel transports ions along the canonical conduction pore. FEBS Lett 581:5024–5028CrossRefPubMedGoogle Scholar
  42. Simon SM, Blobel G (1992) Signal peptides open protein-conducting channels in E. coli. Cell 69:677–684CrossRefPubMedGoogle Scholar
  43. Simon SM, Blobel G, Zimmerberg J (1989) Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli. Proc Natl Acad Sci USA 86:6176–6180CrossRefPubMedGoogle Scholar
  44. Su Z, Zhou X, Loukin SH, Haynes WJ, Saimi Y, Kung C (2009) The use of yeast to understand TRP-channel mechanosensitivity. Pflugers Arch 458:861–867CrossRefPubMedGoogle Scholar
  45. Sutton JM, Lea EJ, Downie JA (1994) The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes. Proc Natl Acad Sci USA 91:9990–9994CrossRefPubMedGoogle Scholar
  46. White PJ, Smahel M, Thiel G (1993) Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers. J Membr Biol 133:145–160PubMedGoogle Scholar
  47. Yellen G (1984) Relief of Na+ block of Ca2+-activated K+ channels by external cations. J Gen Physiol 84:187–199CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Departamento de Biología Molecular de PlantasInstituto de Biotecnología, UNAMCuernavacaMexico
  2. 2.Departamento de FisiologíaBiofísica y Neurociencias. CINVESTAV-IPNMexico CityMexico
  3. 3.Facultad de Medicina, Departamento de FisiologíaUNAM. México. Cd. UniversitariaMéxico, D.F.Mexico
  4. 4.Unidad de Biofísica, CSIC-UPV/EHUUniversidad del País VascoLeioaSpain

Personalised recommendations