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孔通道并介导细胞与细胞通讯的重要方面的蛋白质。人类基因组中有21个连接蛋白基因。这种多样性表明它们是重要的。这一点得到了以下事实的证实，即有许多遗传疾病是由连接蛋白突变引发的，这些疾病共同涵盖了每个主要器官系统。它们能够通过离子和小分子，如三磷酸腺苷（ATP）和葡萄糖。连接蛋白可以以两种模式运作：i）两个相邻细胞中的通道可以对接在一起以形成细胞之间的通道-“间隙连接”;或者ii）它们可以简单地打开到细胞外的空间-“半通道”。通过呼吸调节二氧化碳（CO2）的排泄对生命至关重要。如果过多的二氧化碳在血液中积聚，它就会变成酸性，这可能导致死亡。我们已经发现了证据表明，通过连接蛋白26（Cx 26）的CO2传感对于呼吸的调节很重要。我们已经弄清楚了CO2如何与Cx 26结合，导致半通道打开，并允许ATP释放到细胞外空间。ATP可以扩散并激活附近细胞上的受体。然而，我们最近发现，二氧化碳关闭缝隙连接-通过相同的结合基序，打开半通道。这给了我们一个难题-CO2是通过Cx 26半通道，Cx 26间隙连接还是两者发挥作用？我们目前的遗传工具不能区分这些可能性。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。原始鱼类和两栖类有Cx 26同源物，在蛋白质的C末端有额外的氨基酸（CTT）。这些额外的氨基酸阻止半通道向CO2开放，但不改变差距连接关闭的能力。最令人兴奋的是，当CTT被移植到人Cx 26上以产生嵌合蛋白Cx 26-CTT时，这消除了人Cx 26对CO2的敏感性。Cx 26-CTT亚基有可能成为一种完美的遗传工具，具有从Cx 26半通道去除CO2敏感性的精确选择性，但保留所有其他功能，最重要的是差距连接的CO2敏感性，我们的项目试图记录Cx 26-CTT与野生型（正常）Cx 26的共组装，并表征Cx 26-CTT与野生型（正常）Cx 26的相对比例。CTT与Cx 26亚基在完整的半通道中需要消除其CO2敏感性-这个数字越小，作用越强。我们将通过考虑来自一系列原始鱼类和两栖动物的Cx 26同源物的CTT来优化CTT的效力，以产生共有序列（cCTT）和最小序列（mCTT），并且我们可以串联多个CTT以实现更大的效力。在这项开发工作的最后，我们将在体外Cx 26-CTT去除内源性表达的Cx 26的CO2敏感性和它的选择性之间的半通道和间隙连接和其他相关connexin. Have开发了一个有效的和选择性的工具在体外，我们将移动到显示，这在体内工作，以改变呼吸的敏感性。这将通过设计病毒来实现，这些病毒可以在脑干的非常特定的细胞中引起Cx 26-CTT的表达，我们知道Cx 26在调节呼吸中起作用。我们的项目将开发和验证一套遗传工具，这将实现前所未有的事情：选择性地去除Cx 26半通道的CO2敏感性。这将是其他研究的有力推动者-例如，调查Cx 26半通道如何有助于：整个生命过程中呼吸的控制;大脑中血流的控制以及为什么这会增加到大脑活跃的区域;以及，通过将这些工具释放给其他人，Cx 26的CO2敏感性如何有助于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