Advertisement

Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 388, Issue 9, pp 973–981 | Cite as

The interaction between delayed rectifier channel alpha-subunits does not involve hetero-tetramer formation

  • Peter Biliczki
  • Andre Rüdiger
  • Zenawit Girmatsion
  • Marc Pourrier
  • Aida M. Mamarbachi
  • Terence E. Hébert
  • Ralf P. Brandes
  • Stefan H. Hohnloser
  • Stanley Nattel
  • Joachim R. EhrlichEmail author
Original Article

Abstract

We have previously reported a physiologically relevant interaction between KCNQ1 (Q1) and KCNH2 (H2). While the H2 C-terminus has been suggested to play a role, so far, no more detailed information regarding the interaction site is available. The methods used in the study are cell culture, PCR for mutagenesis, patch clamp for ion current recordings, co-immunoprecipitation for determination of protein interaction. Co-expression of Q1 and H2 resulted in an increase of I H2 (tails after +50 mV; Q1 + H2, 36 ± 6 pA/pF; H2, 14 ± 2 pA/pF; n = 10; 12; P < 0.05). Upon expressing a non-conductive (dominant-negative) Q1-pore mutation (dnQ1), there was still an increase in I H2 (tails after +50 mV; H2 + dnQ1, 24 ± 4 pA/pF; n = 10; P < 0.05) making the pore region unlikely as an interaction site. Experiments using the KCNH2-pore blocking agent quinidine supported these findings. If Q1 and H2 formed hetero-tetramers, steric changes within the pore should change the quinidine half-inhibitory concentrations (IC50). However, I H2 sensitivity did not significantly change in the presence or absence of Q1 (IC50 341 ± 63 vs. 611 ± 293 nmol/L, respectively, P = n.s.), providing further evidence that the pore is not a likely H2-Q1 interaction site. To obtain further insights into the role of intra-cytoplasmic structures, we used both C- and N-terminally truncated mutant H2 proteins. Both H2 mutants co-immunoprecipitated with Q1, suggesting no specific role of C- or N-termini. Accordingly, rather than these, the transmembrane domains of the α-subunits appear relevant for the interaction. Our results largely exclude the formation of hetero-tetramers between H2 and Q1 comprising the pore region or H2 C- or N-termini.

Keywords

Hetero-tetramer KCNQ1 KCNH2 protein interaction 

Notes

Acknowledgments

The expert technical assistance of Sabine Harenkamp and Christin Lößl is gratefully acknowledged.

Sources of funding

Dr. Ehrlich received support from the Deutsche Forschungsgemeinschaft (EH 201/1).

References

  1. Anderson CL, Delisle BP, Anson BD, Kilby JA, Will ML, Tester DJ, Gong Q, Zhou Z, Ackerman MJ, January CT (2006) Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism. Circulation 113:365–373CrossRefPubMedGoogle Scholar
  2. Biliczki P, Girmatsion Z, Brandes RP, Harenkamp S, Pitard B, Charpentier F, Hebert TE, Hohnloser SH, Baro I, Nattel S, Ehrlich JR (2009) Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction. Heart Rhythm 6:1792–1801CrossRefPubMedGoogle Scholar
  3. Chen J, Zou A, Splawski I, Keating MT, Sanguinetti MC (1999) Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of HERG potassium channels accelerate channel deactivation. J Biol Chem 274:10113–10118CrossRefPubMedGoogle Scholar
  4. 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 280:69–77CrossRefPubMedGoogle Scholar
  5. Ehrlich JR, Pourrier M, Weerapura M, Ethier N, Marmabachi AM, Hebert TE, Nattel S (2004) KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents. J Biol Chem 279:1233–1241CrossRefPubMedGoogle Scholar
  6. Ficker E, Obejero-Paz CA, Zhao S, Brown AM (2002) The binding site for channel blockers that rescue misprocessed human long QT syndrome type 2 ether-a-gogo-related gene (HERG) mutations. J Biol Chem 277:4989–4998CrossRefPubMedGoogle Scholar
  7. Ficker E, Dennis AT, Wang L, Brown AM (2003) Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG. Circ Res 92:e87–e100CrossRefPubMedGoogle Scholar
  8. Fink M, Duprat F, Heurteaux C, Lesage F, Romey G, Barhanin J, Lazdunski M (1996) Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K+ channel proteins via their N-terminal end. FEBS Lett 378:64–68CrossRefPubMedGoogle Scholar
  9. Hayashi K, Shuai W, Sakamoto Y, Higashida H, Yamagishi M, Kupershmidt S (2010) Trafficking-competent KCNQ1 variably influences the function of HERG long QT alleles. Heart Rhythm 7:973–980PubMedCentralCrossRefPubMedGoogle Scholar
  10. Hedley PL, Jorgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink PA, Kanters JK, Corfield VA, Christiansen M (2009) The genetic basis of long QT and short QT syndromes: a mutation update. Hum Mutat 30:1486–1511CrossRefPubMedGoogle Scholar
  11. Lees-Miller JP, Kondo C, Wang L, Duff HJ (1997) Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circ Res 81:719–726CrossRefPubMedGoogle Scholar
  12. London B, Trudeau MC, Newton KP, Beyer AK, Copeland NG, Gilbert DJ, Jenkins NA, Satler CA, Robertson GA (1997) Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ Res 81:870–878CrossRefPubMedGoogle Scholar
  13. Melman YF, Krummerman A, McDonald TV (2002) KCNE regulation of KvLQT1 channels: structure-function correlates. Trends Cardiovasc Med 12:182–187CrossRefPubMedGoogle Scholar
  14. Miake J, Marban E, Nuss HB (2003) Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression. J Clin Invest 111:1529–1536PubMedCentralCrossRefPubMedGoogle Scholar
  15. Moss AJ, Zareba W, Kaufman ES, Gartman E, Peterson DR, Benhorin J, Towbin JA, Keating MT, Priori SG, Schwartz PJ, Vincent GM, Robinson JL, Andrews ML, Feng C, Hall WJ, Medina A, Zhang L, Wang Z (2002) Increased risk of arrhythmic events in long-QT syndrome with mutations in the pore region of the human ether-a-go-go-related gene potassium channel. Circulation 105:794–799CrossRefPubMedGoogle Scholar
  16. Neyroud N, Richard P, Vignier N, Donger C, Denjoy I, Demay L, Shkolnikova M, Pesce R, Chevalier P, Hainque B, Coumel P, Schwartz K, Guicheney P (1999) Genomic organization of the KCNQ1 K+ channel gene and identification of C-terminal mutations in the long-QT syndrome. Circ Res 84:290–297CrossRefPubMedGoogle Scholar
  17. Roden DM (2006) Long QT syndrome: reduced repolarization reserve and the genetic link. J Intern Med 259:59–69CrossRefPubMedGoogle Scholar
  18. Sanguinetti MC, Curran ME, Spector PS, Keating MT (1996) Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci U S A 93:2208–2212PubMedCentralCrossRefPubMedGoogle Scholar
  19. Wang Q, Curran ME, Splawski I, Burn TC, Millholland JM, VanRaay TJ, Shen J, Timothy KW, Vincent GM, de Jager T, Schwartz PJ, Toubin JA, Moss AJ, Atkinson DL, Landes GM, Connors TD, Keating MT (1996) Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 12:17–23CrossRefPubMedGoogle Scholar
  20. Warmke JW, Ganetzky B (1994) A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A 91:3438–3442PubMedCentralCrossRefPubMedGoogle Scholar
  21. Weerapura M, Nattel S, Chartier D, Caballero R, Hebert TE (2002) A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link? J Physiol 540:15–27PubMedCentralCrossRefPubMedGoogle Scholar
  22. Zhou Z, Gong Q, Epstein ML, January CT (1998) HERG channel dysfunction in human long QT syndrome. Intracellular transport and functional defects. J Biol Chem 273:21061–21066CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Peter Biliczki
    • 2
  • Andre Rüdiger
    • 2
  • Zenawit Girmatsion
    • 2
  • Marc Pourrier
    • 5
  • Aida M. Mamarbachi
    • 5
  • Terence E. Hébert
    • 4
  • Ralf P. Brandes
    • 3
  • Stefan H. Hohnloser
    • 2
  • Stanley Nattel
    • 5
  • Joachim R. Ehrlich
    • 1
    • 2
    Email author
  1. 1.St. Josefs-HospitalWiesbadenGermany
  2. 2.Division of CardiologyGoethe-UniversityFrankfurtGermany
  3. 3.Institute of Cardiovascular PhysiologyGoethe-UniversityFrankfurtGermany
  4. 4.Department of Pharmacology and TherapeuticsMcGill UniversityMontréalCanada
  5. 5.Montreal Heart InstituteUniversity of MontrealMontrealCanada

Personalised recommendations