Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1997 Nov 3;16(21):6337–6345. doi: 10.1093/emboj/16.21.6337

Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-à-go-go potassium channel.

J Ludwig 1, D Owen 1, O Pongs 1
PMCID: PMC1170240  PMID: 9400421

Abstract

The specific assembly of subunits to oligomers is an important prerequisite for producing functional potassium channels. We have studied the assembly of voltage-gated rat ether-à-go-go (r-eag) potassium channels with two complementary assays. In protein overlay binding experiments it was shown that a 41-amino-acid domain, close to the r-eag subunit carboxy-terminus, is important for r-eag subunit interaction. In an in vitro expression system it was demonstrated that r-eag subunits lacking this assembly domain cannot form functional potassium channels. Also, a approximately 10-fold molar excess of the r-eag carboxy-terminus inhibited in co-expression experiments the formation of functional r-eag channels. When the r-eag carboxy-terminal assembly domain had been mutated, the dominant-negative effect of the r-eag carboxy-terminus on r-eag channel expression was abolished. The results demonstrate that a carboxy-terminal assembly domain is essential for functional r-eag potassium channel expression, in contrast to the one of Shaker-related potassium channels, which is directed by an amino-terminal assembly domain.

Full Text

The Full Text of this article is available as a PDF (463.3 KB).

Selected References

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

  1. Anderson J. A., Huprikar S. S., Kochian L. V., Lucas W. J., Gaber R. F. Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3736–3740. doi: 10.1073/pnas.89.9.3736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brüggemann A., Pardo L. A., Stühmer W., Pongs O. Ether-à-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP. Nature. 1993 Sep 30;365(6445):445–448. doi: 10.1038/365445a0. [DOI] [PubMed] [Google Scholar]
  3. Chen M. L., Hoshi T., Wu C. F. Heteromultimeric interactions among K+ channel subunits from Shaker and eag families in Xenopus oocytes. Neuron. 1996 Sep;17(3):535–542. doi: 10.1016/s0896-6273(00)80185-3. [DOI] [PubMed] [Google Scholar]
  4. Deal K. K., Lovinger D. M., Tamkun M. M. The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing. J Neurosci. 1994 Mar;14(3 Pt 2):1666–1676. doi: 10.1523/JNEUROSCI.14-03-01666.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  6. Hopkins W. F., Demas V., Tempel B. L. Both N- and C-terminal regions contribute to the assembly and functional expression of homo- and heteromultimeric voltage-gated K+ channels. J Neurosci. 1994 Mar;14(3 Pt 1):1385–1393. doi: 10.1523/JNEUROSCI.14-03-01385.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ikeda S. R., Soler F., Zühlke R. D., Joho R. H., Lewis D. L. Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA. Pflugers Arch. 1992 Nov;422(2):201–203. doi: 10.1007/BF00370422. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  10. Lee T. E., Philipson L. H., Kuznetsov A., Nelson D. J. Structural determinant for assembly of mammalian K+ channels. Biophys J. 1994 Mar;66(3 Pt 1):667–673. doi: 10.1016/s0006-3495(94)80840-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Li M., Jan Y. N., Jan L. Y. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. Science. 1992 Aug 28;257(5074):1225–1230. doi: 10.1126/science.1519059. [DOI] [PubMed] [Google Scholar]
  12. Li X., Xu J., Li M. The human delta1261 mutation of the HERG potassium channel results in a truncated protein that contains a subunit interaction domain and decreases the channel expression. J Biol Chem. 1997 Jan 10;272(2):705–708. doi: 10.1074/jbc.272.2.705. [DOI] [PubMed] [Google Scholar]
  13. Liman E. R., Tytgat J., Hess P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron. 1992 Nov;9(5):861–871. doi: 10.1016/0896-6273(92)90239-a. [DOI] [PubMed] [Google Scholar]
  14. Liu D. T., Tibbs G. R., Siegelbaum S. A. Subunit stoichiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function. Neuron. 1996 May;16(5):983–990. doi: 10.1016/s0896-6273(00)80121-x. [DOI] [PubMed] [Google Scholar]
  15. Ludwig J., Terlau H., Wunder F., Brüggemann A., Pardo L. A., Marquardt A., Stühmer W., Pongs O. Functional expression of a rat homologue of the voltage gated either á go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart. EMBO J. 1994 Oct 3;13(19):4451–4458. doi: 10.1002/j.1460-2075.1994.tb06767.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MacKinnon R. Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature. 1991 Mar 21;350(6315):232–235. doi: 10.1038/350232a0. [DOI] [PubMed] [Google Scholar]
  17. Parcej D. N., Dolly J. O. Dendrotoxin acceptor from bovine synaptic plasma membranes. Binding properties, purification and subunit composition of a putative constituent of certain voltage-activated K+ channels. Biochem J. 1989 Feb 1;257(3):899–903. doi: 10.1042/bj2570899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parcej D. N., Scott V. E., Dolly J. O. Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain. Biochemistry. 1992 Nov 17;31(45):11084–11088. doi: 10.1021/bi00160a018. [DOI] [PubMed] [Google Scholar]
  19. Peled-Zehavi H., Arkin I. T., Engelman D. M., Shai Y. Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes. Biochemistry. 1996 May 28;35(21):6828–6838. doi: 10.1021/bi952988t. [DOI] [PubMed] [Google Scholar]
  20. Pfaffinger P. J., DeRubeis D. Shaker K+ channel T1 domain self-tetramerizes to a stable structure. J Biol Chem. 1995 Dec 1;270(48):28595–28600. doi: 10.1074/jbc.270.48.28595. [DOI] [PubMed] [Google Scholar]
  21. Rehm H., Lazdunski M. Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4919–4923. doi: 10.1073/pnas.85.13.4919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rettig J., Heinemann S. H., Wunder F., Lorra C., Parcej D. N., Dolly J. O., Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature. 1994 May 26;369(6478):289–294. doi: 10.1038/369289a0. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schönherr R., Heinemann S. H. Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J Physiol. 1996 Jun 15;493(Pt 3):635–642. doi: 10.1113/jphysiol.1996.sp021410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sentenac H., Bonneaud N., Minet M., Lacroute F., Salmon J. M., Gaymard F., Grignon C. Cloning and expression in yeast of a plant potassium ion transport system. Science. 1992 May 1;256(5057):663–665. doi: 10.1126/science.1585180. [DOI] [PubMed] [Google Scholar]
  26. Sewing S., Roeper J., Pongs O. Kv beta 1 subunit binding specific for shaker-related potassium channel alpha subunits. Neuron. 1996 Feb;16(2):455–463. doi: 10.1016/s0896-6273(00)80063-x. [DOI] [PubMed] [Google Scholar]
  27. Shen N. V., Chen X., Boyer M. M., Pfaffinger P. J. Deletion analysis of K+ channel assembly. Neuron. 1993 Jul;11(1):67–76. doi: 10.1016/0896-6273(93)90271-r. [DOI] [PubMed] [Google Scholar]
  28. Shen N. V., Pfaffinger P. J. Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins. Neuron. 1995 Mar;14(3):625–633. doi: 10.1016/0896-6273(95)90319-4. [DOI] [PubMed] [Google Scholar]
  29. Spector P. S., Curran M. E., Zou A., Keating M. T., Sanguinetti M. C. Fast inactivation causes rectification of the IKr channel. J Gen Physiol. 1996 May;107(5):611–619. doi: 10.1085/jgp.107.5.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stansfeld C. E., Röper J., Ludwig J., Weseloh R. M., Marsh S. J., Brown D. A., Pongs O. Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9910–9914. doi: 10.1073/pnas.93.18.9910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Terlau H., Heinemann S. H., Stühmer W., Pongs O., Ludwig J. Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment. J Physiol. 1997 Aug 1;502(Pt 3):537–543. doi: 10.1111/j.1469-7793.1997.537bj.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Terlau H., Ludwig J., Steffan R., Pongs O., Stühmer W., Heinemann S. H. Extracellular Mg2+ regulates activation of rat eag potassium channel. Pflugers Arch. 1996 Jun;432(2):301–312. doi: 10.1007/s004240050137. [DOI] [PubMed] [Google Scholar]
  33. Tu L., Santarelli V., Sheng Z., Skach W., Pain D., Deutsch C. Voltage-gated K+ channels contain multiple intersubunit association sites. J Biol Chem. 1996 Aug 2;271(31):18904–18911. doi: 10.1074/jbc.271.31.18904. [DOI] [PubMed] [Google Scholar]
  34. Tucker S. J., Bond C. T., Herson P., Pessia M., Adelman J. P. Inhibitory interactions between two inward rectifier K+ channel subunits mediated by the transmembrane domains. J Biol Chem. 1996 Mar 8;271(10):5866–5870. doi: 10.1074/jbc.271.10.5866. [DOI] [PubMed] [Google Scholar]
  35. Warmke J. W., Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3438–3442. doi: 10.1073/pnas.91.8.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Warmke J., Drysdale R., Ganetzky B. A distinct potassium channel polypeptide encoded by the Drosophila eag locus. Science. 1991 Jun 14;252(5012):1560–1562. doi: 10.1126/science.1840699. [DOI] [PubMed] [Google Scholar]
  37. Wu C. F., Ganetzky B., Haugland F. N., Liu A. X. Potassium currents in Drosophila: different components affected by mutations of two genes. Science. 1983 Jun 3;220(4601):1076–1078. doi: 10.1126/science.6302847. [DOI] [PubMed] [Google Scholar]
  38. Xu J., Yu W., Jan Y. N., Jan L. Y., Li M. Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between the alpha-subunits. J Biol Chem. 1995 Oct 20;270(42):24761–24768. doi: 10.1074/jbc.270.42.24761. [DOI] [PubMed] [Google Scholar]
  39. Yu W., Xu J., Li M. NAB domain is essential for the subunit assembly of both alpha-alpha and alpha-beta complexes of shaker-like potassium channels. Neuron. 1996 Feb;16(2):441–453. doi: 10.1016/s0896-6273(00)80062-8. [DOI] [PubMed] [Google Scholar]
  40. Zhong Y., Wu C. F. Alteration of four identified K+ currents in Drosophila muscle by mutations in eag. Science. 1991 Jun 14;252(5012):1562–1564. doi: 10.1126/science.2047864. [DOI] [PubMed] [Google Scholar]
  41. Zhong Y., Wu C. F. Modulation of different K+ currents in Drosophila: a hypothetical role for the Eag subunit in multimeric K+ channels. J Neurosci. 1993 Nov;13(11):4669–4679. doi: 10.1523/JNEUROSCI.13-11-04669.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES