Biliczki Peter, Rüdiger Andre, Girmatsion Zenawit, Pourrier Marc, Mamarbachi Aida M, Hébert Terence E, Brandes Ralf P, Hohnloser Stefan H, Nattel Stanley, Ehrlich Joachim R
Division of Cardiology, Goethe-University, Theodor Stern Kai 7, 60590, Frankfurt, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2015 Sep;388(9):973-81. doi: 10.1007/s00210-015-1108-3. Epub 2015 Mar 20.
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.
我们之前报道过KCNQ1（Q1）和KCNH2（H2）之间存在生理相关的相互作用。虽然有人提出H2的C末端发挥了作用，但到目前为止，关于相互作用位点尚无更详细的信息。该研究中使用的方法包括细胞培养、用于诱变的PCR、用于离子电流记录的膜片钳以及用于确定蛋白质相互作用的免疫共沉淀。Q1和H2的共表达导致I H2增加（在 +50 mV后的尾电流；Q1 + H2，36±6 pA/pF；H2，14±2 pA/pF；n = 10；12；P < 0.05）。在表达非导电（显性负性）的Q1孔突变体（dnQ1）后，I H2仍有增加（在 +50 mV后的尾电流；H2 + dnQ1，24±4 pA/pF；n = 10；P < 0.05），这使得孔区域不太可能是相互作用位点。使用KCNH2孔阻断剂奎尼丁进行的实验支持了这些发现。如果Q1和H2形成异源四聚体，孔内的空间变化应该会改变奎尼丁的半抑制浓度（IC50）。然而，在有或没有Q1的情况下，I H2敏感性没有显著变化（IC50分别为341±63与611±293 nmol/L，P = 无显著差异），这进一步证明孔不太可能是H2 - Q1相互作用位点。为了进一步深入了解胞质内结构的作用，我们使用了C末端和N末端截短的突变体H2蛋白。两种H2突变体都与Q1进行了免疫共沉淀，表明C末端或N末端没有特定作用。因此，α亚基的跨膜结构域而非这些末端似乎与相互作用相关。我们的结果在很大程度上排除了H2和Q1之间形成包含孔区域或H2 C末端或N末端的异源四聚体的可能性。
