Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K+ channel, Tok1p

  • Anja Roller
  • Gabriel Natura
  • Hermann Bihler
  • Clifford L. Slayman
  • Adam BertlEmail author
Ion Channels


Tandem pore-loop potassium channels differ from the majority of K+ channels in that a single polypeptide chain carries two K+-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP1M–MP2M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P1 and Y424 in P2) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P1 mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P1 than at P2 appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P2 (overwhelmingly TIGYGD) to be much stabler than that of P1 (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)—which inverts the order of residues from the wild-type channel—reduces the expressed channel conductance by a large factor (20-fold, cf. <twofold for the single mutants).


Voltage dependence Conductance Potassium channel Whole-cell recording Activation kinetics 



The authors are indebted to Drs. T. Miosga and P. Ljungdahl for yeast strains. The work was supported by Grants from the Deutsche Forschungsgemeinschaft (to A.B.) and from the US National Institutes of Health (GM-60696, to C.L.S.).


  1. 1.
    Bertl A, Slayman CL (1993) Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies. J Exp Biol 172:271–287Google Scholar
  2. 2.
    Bertl A, Bihler H, Kettner C, Slayman CL (1998a) Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae. Pflügers Arch 436:999–1013PubMedCrossRefGoogle Scholar
  3. 3.
    Bertl A, Bihler H, Reid JD, Scheurmann-Kettner C, Slayman CL (1998b) Physiological characterization of the yeast plasma-membrane outward rectifying K+ channel, DUK1 (TOK1) in situ. J Membr Biol 162:67–80PubMedCrossRefGoogle Scholar
  4. 4.
    Bertl A, Ramos J, Ludwig J, Lichtenberg-Frate H, Reid JD, Bihler H, Calero F, Martinez P, Ljungdahl PO (2003) Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2, and tok1 null mutations. Mol Microbiol 47:767–780PubMedCrossRefGoogle Scholar
  5. 5.
    Bihler H, Slayman CL, Bertl A (2002) Low-affinity potassium uptake by Saccharomyces cerevisiae is mediated by NSC1, a calcium-blocked non-specific cation channel. Biochim Biophys Acta 1558:109–118PubMedCrossRefGoogle Scholar
  6. 6.
    Chapman ML, Krovetz HS, VanDongen AMJ (2001) GYGD pore motifs in neighbouring potassium channel subunits interact to determine ion selectivity. J Physiol 530:21–33PubMedCrossRefGoogle Scholar
  7. 7.
    Conway EJ, Breen J (1945) An ‘ammonia’-yeast and some of its properties. Biochem J 39:368–371PubMedGoogle Scholar
  8. 8.
    Cordero-Morales JF, Cuello LG, Zhao Y-X, Jogini V, Cortes DM, Roux B, Perozo E (2006) Molecular determinants of gating at the potassium-channel selectivity filter. Nature Str Mol Biol 13:311–318CrossRefGoogle Scholar
  9. 9.
    Doyle DA, Morais-Cabral J, 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 280:69–77PubMedCrossRefGoogle Scholar
  10. 10.
    Durell SR, Guy HR (1999) Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA channel. Biophys J 77:789–807PubMedCrossRefGoogle Scholar
  11. 11.
    Durell SR, Hao Y, Guy HR (1998) Structural models of the transmembrane region of voltage-gated and other K+ channels in open, closed, and inactivated conformations. J Struc Biol 121:263–284CrossRefGoogle Scholar
  12. 12.
    Durell SR, Shrivastava IH, Guy HR (2004) Models of the structure and voltage-gating mechanism of the Shaker K+ channel. Biophys J 87:2116–2130PubMedCrossRefGoogle Scholar
  13. 13.
    Fairman C, Zhou X, Kung C (1999) Potassium uptake through the TOK1 K+ channel in the budding yeast. J Membr Biol 168:149–157PubMedCrossRefGoogle Scholar
  14. 14.
    Gray AT, Winegar BD, Leonoudakis DJ, Forsayeth JR, Yost CS (1998) TOK1 is a volatile anesthetic stimulated K+ channel. Anesthesiology 88:1076–1084PubMedCrossRefGoogle Scholar
  15. 15.
    Heginbotham L, Lu Z, Abramson T, MacKinnon R (1994) Mutations in the K+ channel signature sequence. Biophys J 66:1061–1067PubMedGoogle Scholar
  16. 16.
    Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167PubMedCrossRefGoogle Scholar
  17. 17.
    Jan LY, Jan YN (1997) Cloned potassium channels from eukaryotes and prokaryotes. Annu Rev Neurosci 20:91–123PubMedCrossRefGoogle Scholar
  18. 18.
    Jiang Y-X, Lee A, Chen J-Y, Ruta V, Cadene M, Chait BT, MacKinnon R (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423:33–41PubMedCrossRefGoogle Scholar
  19. 19.
    Johansson I, Blatt MR (2006) Interactive domains between pore loops of the yeast K+ channel TOK1 associate with extracellular K+ sensitivity. Biochem J 393:645–655PubMedCrossRefGoogle Scholar
  20. 20.
    Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK, Goldstein SAN (1995) A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376:690–695PubMedCrossRefGoogle Scholar
  21. 21.
    Ko CH, Gaber FR (1991) TRKI and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol Cell Biol 11:4266–4273PubMedGoogle Scholar
  22. 22.
    Kuroda T, Bihler H, Bashi E, Slayman CL, Rivetta A (2004) Currents through the yeast TRK-potassium transporters, measured by patch-clamping, are dominated by chloride and depend upon strain background. J Membr Biol 198:177–192PubMedCrossRefGoogle Scholar
  23. 23.
    Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, Romey G, Barhanin J (1996) A pH-sensitive yeast outward rectifier K+ channel with two pore domains and novel gating properties. J Biol Chem 271:4183–4187PubMedCrossRefGoogle Scholar
  24. 24.
    Lopes CM, Gallagher PG, Buck ME, Butler MH, Goldstein SA (2001) Block of Kcnk3 by protons. Evidence that 2-P-domain potassium channel subunits function as homodimers. J Biol Chem 276:24449–24452PubMedCrossRefGoogle Scholar
  25. 25.
    Loukin SH, Vaillant B, Zhou X-L, Spalding EP, Kung C, Saimi Y (1997) Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1. EMBO J 16:4817–4825PubMedCrossRefGoogle Scholar
  26. 26.
    Miosga T, Witzel A, Zimmermann FK (1994) Sequence and function analysis of a 9.46 kb fragment of Saccharomyces cerevisiae chromosome X. Yeast 10:965–973PubMedCrossRefGoogle Scholar
  27. 27.
    Morais-Cabral JH, Zhou Y-F, MacKinnon R (2001) Energetic optimization of ion conduction rate by the K+ selectivity filter. Nature 414:37–42PubMedCrossRefGoogle Scholar
  28. 28.
    Reid JD, Lukas W, Shafaatian R, Bertl A, Scheurmann-Kettner C, Guy HR, North RA (1996) The S. cerevisiae outwardly-rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains. Recept Channels 4:51–62PubMedGoogle Scholar
  29. 29.
    Roberts SK (2003) TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus. Eukaryot Cell 2:181–90PubMedCrossRefGoogle Scholar
  30. 30.
    Rodriguez-Navarro A, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159:940–945PubMedGoogle Scholar
  31. 31.
    Roller A, Natura G, Bihler H, Slayman CL, Eing C, Bertl A (2005) In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance. Pflügers Arch 451:362–370PubMedCrossRefGoogle Scholar
  32. 32.
    Vergani P, Blatt MR (1999) Mutations in the yeast two-pore K+ channel YKC1 identify functional differences between the pore domains. FEBS Lett 458:285–291PubMedCrossRefGoogle Scholar
  33. 33.
    Véry AA, Sentenac H (2002) Cation channels in the Arabidopsis plasma membrane. Trends Plant Sci 7:168–175PubMedCrossRefGoogle Scholar
  34. 34.
    Yuill KH, Ashmole I, Stanfield PR (2004) The tandem-pore K+ channel TASK-1: mutations of GYG in P1 and GFG in P2 alter ionic selectivity. Biophys J 86(1):549A–549AGoogle Scholar
  35. 35.
    Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001) Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 Å resolution. Nature 414:43–48PubMedCrossRefGoogle Scholar
  36. 36.
    Zhou M, MacKinnon R (2004) A mutant KcsA K+ channel with altered conduction properties and selectivity filter ion distribution. J Mol Biol 338:839–846PubMedCrossRefGoogle Scholar
  37. 37.
    Zhou XL, Vaillant B, Loukin SH, Kung C, Saimi Y (1995) YKC1 encodes the depolarization-activated K+ channel in the plasma membrane of yeast. FEBS Lett 373:170–176PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Anja Roller
    • 1
  • Gabriel Natura
    • 2
  • Hermann Bihler
    • 1
  • Clifford L. Slayman
    • 3
  • Adam Bertl
    • 1
    • 4
    Email author
  1. 1.Botanisches Institut IUniversität Karlsruhe (TH)KarlsruheGermany
  2. 2.Physiologisches InstitutUniversität JenaJenaGermany
  3. 3.Department of Cellular and Molecular PhysiologyYale School of MedicineNew HavenUSA
  4. 4.Botanisches Institut, Fachbereich BiologieTU DarmstadtDarmstadtGermany

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