title: The interaction between delayed rectifier channel alpha-subunits does not involve hetero-tetramer formation.
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.
authors: Division of Cardiology, Goethe-University, Theodor Stern Kai 7, 60590, Frankfurt, Germany.
authors: Peter
authors: P
authors: Biliczki
authors: Andre
authors: A
authors: Rüdiger
authors: Zenawit
authors: Z
authors: Girmatsion
authors: Marc
authors: M
authors: Pourrier
authors: Aida M
authors: AM
authors: Mamarbachi
authors: Terence E
authors: TE
authors: Hébert
authors: Ralf P
authors: RP
authors: Brandes
authors: Stefan H
authors: SH
authors: Hohnloser
authors: Stanley
authors: S
authors: Nattel
authors: Joachim R
authors: JR
authors: Ehrlich
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D020128:Inhibitory Concentration 50; D051657:KCNQ1 Potassium Channel / Q000737:chemistry / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011802:Quinidine / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D051657:KCNQ1 Potassium Channel; D011802:Quinidine