Gating and Regulation of Connexin Hemichannels

在20种人类连接蛋白亚型中，绝大多数与病理有关 功能障碍，这表明这些蛋白质在人类健康中发挥着重要的生理作用。 尽管连接蛋白通道对原子离子和小分子代谢物都具有通透性，但它们 对不同的分子具有高度的选择性，这可能决定了它们不同的生物学功能。 它们还受到各种刺激的调节，包括钙离子、电压和pH。我们的理解是， 然而，渗透和门控背后的分子机制是非常肤浅的， 特别是与其他经典离子通道相比。最近，我们做出了令人惊讶的事情 连接蛋白半通道(和其他大孔道)不是被动扩散的发现 分子的小孔。相反，它们显示饱和，表明渗透中有结合部位。 可能以与转运体/载体类似的方式决定选择性的途径。另外， 我们实验室发表的和初步的数据表明，N末端(NT)起着重要作用 在分子的渗透和孔道堵塞中，排列在孔道孔道内的区域。因此，我们 假设连接蛋白半管中的NT起：(1)作为一种关键的能量屏障 分子的传输和(2)作为控制离子传导的门。在这项提案中，我们 关注两个连接蛋白，Cx26和Cx30，在这两个连接蛋白中，人类在NT上的突变与 耳聋和先天性皮肤病。我们将确定这些NT突变是否会影响 通透性、门控或两者兼而有之，并将提供半隧道功能障碍的机制基础。 已经解决了连接蛋白通道的新的高分辨率结构，这将作为 建议的分子动力学模拟指南。此外，我们还将补充 具有实验研究的计算数据，以解决仍然存在的关键机制问题 仍无人接听。我们的提议意义重大，因为我们正在测试一种创新的 假说，将提供更好的理解渗透和门控的机制 连接蛋白通道。重要的是，这将有助于合理开发和设计药物或 其他治疗方法，可以特别纠正或补偿不同的人类 由连接蛋白功能障碍引起的疾病，包括耳聋和皮肤病。

The vast majority of the 20 human connexin isoforms have been associated with pathological dysfunctions, suggesting an essential physiological role for these proteins in human health. Although connexin channels are permeable to both atomic ions and small metabolites, they are highly selective to different molecules, which likely determines their distinct biological functions. They are also regulated by various stimuli, including Ca2+, voltage, and pH. Our understanding, however, of the molecular mechanisms underlying permeation and gating is extremely superficial, especially when compared with other classical ion channels. Recently, we made the surprising discovery that connexin hemichannels (and other large-pore channels) are not passive diffusion pores for molecules. Instead, they display saturation suggesting binding sites in the permeation pathway that might determine selectivity in a similar manner to transporters/carriers. Additionally, published and preliminary data from our lab suggested a major role for the N-terminal (NT) domain, which lines the channel pore, in permeation of molecules and pore occlusion. Thus, we hypothesize that the NT in connexin hemichannels serves: (1) as a critical energetic barrier for transport of molecules and (2) as the gate for controlling ionic conduction. In this proposal, we focus on two connexins, Cx26 and Cx30, in which human mutations at the NT are associated with deafness and congenital skin disorders. We will identify whether these NT mutations affect permeability, gating or both, and will provide a mechanistic basis of hemichannel dysfunction. Novel high-resolution structures of the connexin channel have been solved, which will serve as a guide for the proposed molecular dynamics simulations. Furthermore, we will complement computational data with experimental studies addressing key mechanistic questions that remain still unanswered. Our proposal is highly significant because we are testing an innovative hypothesis that will provide better understanding of the mechanisms of permeation and gating of connexin channels. Importantly, this will assist in the rational development and design of drugs or other therapeutic approaches that can specifically correct or compensate for the varied human diseases produced by connexin protein dysfunction, including deafness and skin disorders.