New tools for investigating connexin26 hemichannel function in physiological systems

研究生理系统中 connexin26 半通道功能的新工具

• 批准号: BB/T013346/1
• 负责人: Nicholas Dale
• 金额: $ 87.53万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT013346%2F1
• 关键词: New; tools; investigating; connexin26; hemichannel

Connexins are proteins that form large-pored channels in the cell membrane and mediate important aspects of cell to cell communication. There are 21 connexin genes in the human genome. This multiplicity shows that they are important. This is confirmed by the fact that there are many genetic diseases conditions that are triggered by mutations of connexins and these diseases collectively encompass every major organ system. They are capable of passing ions and small molecules such as adenosine triphosphate (ATP) and glucose. Connexins can operate in two modes: i) channels in two adjacent cells can dock together to form a passageway between the cells -a "gap junction"; or ii) they can simply open into the space outside the cell -a "hemichannel".Regulated excretion of carbon dioxide (CO2) via breathing is vital for life. If too much CO2 builds up in the blood it becomes acidic and this can cause death. We have developed evidence that CO2-sensing via Connexin26 (Cx26) is important for the regulation of breathing. We have worked out how CO2 binds to Cx26 to cause the hemichannel to open and allow release of ATP into the extracellular space. ATP can diffuse and activate receptors on nearby cells. However, we have recently found that CO2 closes gap junctions -via the same binding motif that opens hemichannels. This gives us a conundrum -does CO2 exert its action via Cx26 hemichannels, Cx26 gap junctions, or both? Our current genetic tools do not discriminate between these possibilities. Our recent discoveries in primitive fish and mammals enable us to address this question. Primitive fish and amphibia have Cx26 homologues with extra amino acids on the C-terminus of the protein (CTT). These extra amino acids prevent the hemichannel from opening to CO2 but do not alter the ability of the gap junction to close. Most excitingly, when the CTT is grafted onto human Cx26 to make a chimaeric protein, Cx26-CTT, this removes CO2 sensitivity from human Cx26. The Cx26-CTT subunit has the potential to be a perfect genetic tool with exquisite selectivity for removing CO2-sensitivity from the Cx26 hemichannel, but leaving all other functions, most importantly the CO2-sensitivity of the gap junction, unaltered.Our project seeks to document the coassembly of Cx26-CTT with wild type (normal) Cx26 and characterize the relative proportion of Cx26-CTT vs Cx26 subunits in the completed hemichannel required to remove its CO2 sensitivity -the smaller this number the more potent the action. We shall optimize the potency of the CTT by taking into account the CTTs of Cx26 homologues from a range of primitive fish and amphibia to produce a consensus sequence (cCTT) and minimal sequence (mCTT), and we may concatenate multiple CTTs to achieve greater potency. At the end of this development work we will characterize in vitro the efficacy of Cx26-CTT in removing CO2-sensitivity from endogenously expressed Cx26 and its selectivity between hemichannels and gap junctions and between other related connexins.Having developed a potent and selective tool in vitro, we shall move to show that this works in vivo to alter the sensitivity of breathing. This will be achieved by designing viruses that can cause expression of Cx26-CTT in very specific cells of the brain stem in which we know Cx26 plays a role in regulating breathing.Our project will develop and validate a set of genetic tools that will accomplish something unprecedented: selective removal of CO2 sensitivity from Cx26 hemichannels. This will be a powerful enabler of other research -for example to investigate how Cx26 hemichannels contribute to: the control of breathing throughout the entire life course; the control of blood flow in the brain and why this is increased to areas of the brain that are active; and, by releasing these tools to others, how the CO2-sensitivity of Cx26 contributes to the physiology of other organ systems.

连接蛋白是在细胞膜中形成大型通道的蛋白质，并介导细胞的重要方面到细胞通信。人基因组中有21种连接蛋白基因。这种多重性表明它们很重要。这一事实证实了这一事实，即连接蛋白突变和这些疾病共同涵盖了每个主要器官系统，这些遗传疾病疾病会引起。它们能够通过离子和小分子，例如三磷酸腺苷（ATP）和葡萄糖。连接蛋白可以以两种模式运行：i）两个相邻单元中的通道可以停靠在一起以形成一个通道-A“间隙连接”；或ii）它们可以简单地进入单元格 - A“半通道”外的空间。通过呼吸调节二氧化碳（CO2）的排泄对生命至关重要。如果在血液中积聚了太多的二氧化碳，它就会变得酸性，这可能导致死亡。我们已经开发了证据表明，通过连接蛋白26（CX26）对二氧化碳感应对于调节呼吸很重要。我们已经计算出二氧化碳如何与CX26结合，以使半通道打开并允许ATP释放到细胞外空间中。 ATP可以在附近细胞上扩散和激活受体。但是，我们最近发现，CO2关闭了差距连接 - 视频与打开半通道相同的结合基序。这使我们有一个难题 - 二氧化碳通过CX26半通道，CX26间隙连接或两者兼而有之。我们当前的遗传工具不会区分这些可能性。我们最近在原始鱼类和哺乳动物中的发现使我们能够解决这个问题。原始鱼类和两栖动物的CX26同源物在蛋白质的C末端（CTT）上具有额外的氨基酸。这些额外的氨基酸防止半通道开放到二氧化碳，但不会改变间隙连接关闭的能力。最令人兴奋的是，当将CTT移植到人CX26上以生成嵌合蛋白CX26-CTT时，这会消除人CX26的CO2敏感性。 CX26-CTT亚基有可能成为一个完美的遗传工具，具有从CX26 Hemichannel中删除CO2敏感性的精确选择性，但是留下所有其他功能，最重要的是，GAP连接的CO2敏感性，未更改的co2敏感性。消除其二氧化碳灵敏度所需的完整半通道中的CX26 -CTT与CX26亚基 - 该数字越小，动作越有效。我们应考虑到来自原始鱼类和两栖动物的CX26同源物的CTT来优化CTT的效力，以产生共识序列（CCTT）和最小序列（MCTT），并且我们可能会综合多个CTT，以实现更大的CTT。 At the end of this development work we will characterize in vitro the efficacy of Cx26-CTT in removing CO2-sensitivity from endogenously expressed Cx26 and its selectivity between hemichannels and gap junctions and between other related connexins.Having developed a potent and selective tool in vitro, we shall move to show that this works in vivo to alter the sensitivity of breathing.这将通过设计可能导致CX26-CTT表达的病毒在脑干的非常特定的细胞中表达，我们知道CX26在调节呼吸中起着作用。您的项目将开发和验证一组遗传工具，这些工具将完成一套无预示的东西：选择性地去除COX敏感性从CX26 Hemichelels中敏感。这将是其他研究的强大推动力 - 例如，研究CX26半通道如何贡献：整个生命过程中呼吸的控制；控制大脑的血液流动以及为什么将其增加到活跃的大脑区域；而且，通过将这些工具释放给其他工具，CX26的二氧化碳敏感性如何促进其他器官系统的生理学。

项目成果

And they're out of the gate.

他们已经出了门了。

• DOI: 10.1113/jp281784
• 发表时间: 2021
• 期刊: The Journal of physiology
• 作者: Dale N