Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

中文翻译：

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AFM 成像揭示连接蛋白 26/30 异聚半通道的多分散分子结构。

连接蛋白（Cx）蛋白形成半通道和间隙连接通道，在人类生理和疾病中发挥着多样而深远的作用。间隙连接是由相邻细胞的两个半通道对接形成的细胞间通道阵列。每个六聚半通道包含相同或不同的 Cx 同工型。尽管同聚 Cx 形式已在功能和结构上得到了广泛描述，但异聚 Cx 通道的化学计量和排列仍然未知。然而，后者在人体组织中广泛表达，并且变异可能对通道功能产生重要影响。考虑到大量的潜在亚基排列和化学计量，即使仅组合两种 Cx 亚型，研究异聚 Cx 通道的特性也具有挑战性。为了解决这个问题，我们将 HA 标签设计到 Cx26 或 Cx30 亚基上，并通过原子力显微镜对由 Fab 表位抗体片段配体的半通道进行成像。对于 Cx26-HA/Cx30 或 Cx30-HA/Cx26 异聚通道，Fab-HA 结合分布是二项式的，最多结合三个 Fab-HA。此外，由 Fab-HA 配体的成像 Cx26/Cx30-HA 三重显示出可以从总概率定律推导出来的多种排列。Cx26/Cx30-HA 半通道环状结构的原子力显微镜成像证实了这些发现，并且还检测到化学计量的多分散分布。我们的结果表明，主要亚基化学计量为 3Cx26:3Cx30，其中最丰富的亚基排列为 Cx26-Cx26-Cx30-Cx26-Cx30-Cx30。据我们所知，