Bocksteins Elke, Mayeur Evy, Van Tilborg Abbi, Regnier Glenn, Timmermans Jean-Pierre, Snyders Dirk J
Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
PLoS One. 2014 Jun 5;9(6):e98960. doi: 10.1371/journal.pone.0098960. eCollection 2014.
The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-terminus, but unexpectedly also with the Kv3.1 N-terminus. We confirmed this interaction by Fluorescence Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N-terminal Kv3.1 and Kv6.4 fragments. However, full-length Kv3.1 and Kv6.4 subunits do not form heterotetramers at the plasma membrane. Therefore, additional interactions between the Kv6.4 and Kv2.1 subunits should be important in the Kv2.1/Kv6.4 subfamily-specificity. Using FRET and co-IP approaches with N- and C-terminal fragments we observed that the Kv6.4 C-terminus physically interacts with the Kv2.1 N-terminus but not with the Kv3.1 N-terminus. The N-terminal amino acid sequence CDD which is conserved between Kv2 and KvS subunits appeared to be a key determinant since charge reversals with arginine substitutions abolished the interaction between the N-terminus of Kv2.1 and the C-terminus of both Kv2.1 and Kv6.4. In addition, the Kv6.4(CKv3.1) chimera in which the C-terminus of Kv6.4 was replaced by the corresponding domain of Kv3.1, disrupted the assembly with Kv2.1. These results indicate that the subfamily-specific Kv2.1/Kv6.4 heterotetramerization is determined by interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini in which the conserved N-terminal CDD sequence plays a key role.
“沉默”的电压门控钾离子（KvS）通道亚基Kv6.4在质膜上不能形成具有电功能的同四聚体，而是与Kv2.1亚基组装，生成功能性的Kv2.1/Kv6.4异四聚体。N端T1结构域通过阻止属于不同亚家族的亚基之间的相互作用来决定Kv1-4亚基的亚家族特异性组装。对于Kv6.4，酵母双杂交实验表明Kv6.4的N端与Kv2.1的N端相互作用，但出乎意料的是也与Kv3.1的N端相互作用。我们通过荧光共振能量转移（FRET）和使用N端Kv3.1和Kv6.4片段的共免疫沉淀（co-IP）证实了这种相互作用。然而，全长的Kv3.1和Kv6.4亚基在质膜上不能形成异四聚体。因此，Kv6.4和Kv2.1亚基之间的其他相互作用在Kv2.1/Kv6.4亚家族特异性中应该很重要。使用FRET和co-IP方法结合N端和C端片段，我们观察到Kv6.4的C端与Kv2.1的N端发生物理相互作用，但不与Kv3.1的N端相互作用。Kv2和KvS亚基之间保守的N端氨基酸序列CDD似乎是一个关键决定因素，因为用精氨酸替代进行电荷反转消除了Kv2.1的N端与Kv2.1和Kv6.4的C端之间的相互作用。此外，Kv6.4（CKv3.1）嵌合体（其中Kv6.4的C端被Kv3.1的相应结构域取代）破坏了与Kv2.1的组装。这些结果表明，亚家族特异性的Kv2.1/Kv6.4异四聚体化是由Kv2.1和Kv6.4之间涉及N端和C端的相互作用决定的，其中保守的N端CDD序列起关键作用。
