Centro Interdisciplinario de Neurociencias de Valparaíso, Departamento de Neurociencias, Universidad de Vaparaíso, Valparaíso 2360102, Chile.
Biochem J. 2011 May 15;436(1):35-43. doi: 10.1042/BJ20110008.
Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.
连接子的寡聚化是间隙连接通道形成的关键步骤。连接子家族的某些成员可以与其他成员寡聚化形成功能性异源半通道[例如 Cx43（连接子 43）和 Cx45]，但其他成员则不兼容（例如 Cx43 和 Cx26）。为了找到对寡聚化重要的连接子结构域，我们构建了 Cx43 和 Cx26 之间的嵌合体，并研究了它们与野生型 Cx43、Cx45 或 Cx26 寡聚化的能力。用免疫荧光法进行亚细胞定位、细胞间微注射 Lucifer yellow 的扩散（功能测定）和蔗糖梯度沉降速度（生化测定）分析，检测共表达 Cx43、Cx45 或 Cx26 和单个嵌合体构建体的 HeLa 细胞中嵌合体与野生型连接子之间的相互作用。基于共定位、细胞间通讯的显性负抑制作用以及改变的沉降速度，所有包含 Cx43 的第三跨膜结构域的嵌合体均与野生型 Cx43 相互作用。同样的嵌合体也与共表达的 Cx45 相互作用。相比之下，免疫荧光和示踪剂的细胞内扩散表明，当与 Cx26 共表达时，其他结构域中的氨基酸可能会影响寡聚化的兼容性。总之，这些结果表明，第三跨膜结构域中的氨基酸对于与 Cx43 和 Cx45 的寡聚化至关重要。然而，不同结构域中的基序可能决定了不同连接子亚家族成员的寡聚化兼容性。
