Laboratory of Molecular and Cellular Signaling, Department Cellular and Molecular Medicine, KU Leuven, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000, Louvain, Belgium.
Physiology Group, Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium.
Cell Mol Life Sci. 2018 Jun;75(11):2059-2073. doi: 10.1007/s00018-017-2722-7. Epub 2017 Dec 7.
Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca concentration ([Ca]) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca], which may be involved in pathological hemichannel opening.
间隙连接蛋白 43（Cx43）半通道通过释放 ATP 和其他分子建立局部信号网络，但它们的过度开放可能导致细胞死亡。因此，Cx43 半通道的活性应该受到严格控制。这涉及到 C 端尾部（CT）和细胞质环（CL）之间的相互作用，更具体地说是 CL 内的 L2 结构域。以前的工作通过靶向 L2 结构域，揭示了 Cx43 CT 的最后九个氨基酸的重要作用，因为这九个氨基酸足以恢复 CT 截断的 Cx43 半通道的活性。然而，我们发现 CT 的最后 19 个氨基酸的缺失仅部分降低了与 L2 结构域的结合，表明 CT 中存在第二个 L2 结合区。我们在这里提供的证据表明，SH3 结合域是另一个靶向 L2 结构域的 CT 区域。在功能水平上，SH3 结合域能够恢复 CT 截断的 Cx43 半通道的活性，并减轻高细胞内 Ca 浓度 ([Ca]) 对全长 Cx43 半通道的抑制作用，这一点通过包括对单位 Cx43 半通道活性进行膜片钳研究在内的各种方法得到证明。最后，我们表明在全长 Cx43 半通道中，SH3 结合域或 CT9 区域的缺失均可抑制半通道活性，而两个区域的缺失则完全消除半通道活性。这些结果表明，Cx43 SH3 结合域除了 CT9 区域外，还在高 [Ca] 下对半通道活性进行严格控制，这可能与病理性半通道开放有关。
