Department of Pharmacology and Physiology, Université de Montréal, Canada.
Institute of Structural and Molecular Biology, Birkbeck, University of London, UK.
FEBS Lett. 2022 Mar;596(6):772-783. doi: 10.1002/1873-3468.14279. Epub 2022 Jan 19.
Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the 'T1-chimera' construct of the NaChBac sodium channel by truncating its C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N terminus. Purified T1-chimera channels were tetrameric, conducted Na when reconstituted into proteoliposomes, and were functionally blocked by the drug mibefradil. Both the T1-chimera and full-length NaChBac had comparable expression levels in the membrane, whereas a NaChBac mutant lacking a cytoplasmic domain had greatly reduced membrane expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerisation. This phenomenon is found with other channels, and thus, our findings substantiate this as a common assembly mechanism.
细胞质结构域通常促进多聚体离子通道的功能组装。为了研究这一过程的结构决定因素，我们通过截断 NaChBac 钠通道的 C 末端结构域，并将 Kv1.2 通道的 T1 四聚化结构域拼接至 N 末端，生成了“T1 嵌合体”构建体。纯化的 T1 嵌合体通道为四聚体，在重新构建到脂质体中时可传导 Na+，并被药物米贝地尔功能性阻断。T1 嵌合体和全长 NaChBac 在膜中的表达水平相当，而缺乏细胞质结构域的 NaChBac 突变体的膜表达则大大降低。我们的发现支持这样一种模型，即通过使跨膜区域紧密接近，能够使它们形成四聚体。这种现象在其他通道中也有发现，因此，我们的发现证实了这是一种常见的组装机制。
