New tools for investigating connexin26 hemichannel function in physiological systems

连接蛋白是一种在细胞膜上形成大孔道的蛋白质，在细胞与细胞之间的通讯中起重要的调节作用。在人类基因组中有21个连接蛋白基因。这种多样性表明它们是重要的。事实证实了这一点，即有许多遗传性疾病是由连接蛋白的突变引发的，这些疾病共同涵盖了每个主要的器官系统。它们能够传递离子和小分子，如三磷酸腺苷(ATP)和葡萄糖。连接蛋白可以在两种模式下工作：i)两个相邻细胞中的通道可以对接在一起，形成细胞之间的通道-“缝隙连接”；或者ii)它们可以简单地打开进入细胞外的空间-一个“半通道”。通过呼吸调节二氧化碳(CO2)的排泄对生命至关重要。如果血液中积累了太多的二氧化碳，它就会变成酸性，这可能会导致死亡。我们已经有证据表明，通过连接蛋白26(Cx26)感知二氧化碳对呼吸的调节很重要。我们已经弄清楚了二氧化碳是如何与Cx26结合导致半通道打开并允许ATP释放到细胞外空间的。三磷酸腺苷可以扩散和激活附近细胞上的受体。然而，我们最近发现，二氧化碳通过打开半通道的相同结合基序关闭缝隙连接。这给了我们一个难题--二氧化碳是通过Cx26半通道发挥作用，还是通过Cx26缝隙连接发挥作用，还是两者都有？我们目前的遗传工具并不区分这些可能性。我们最近在原始鱼类和哺乳动物上的发现使我们能够解决这个问题。原始鱼类和两栖动物都有Cx26同源物，在蛋白的C-末端有额外的氨基酸(CTT)。这些额外的氨基酸阻止半通道在二氧化碳下打开，但不会改变缝隙连接关闭的能力。最令人兴奋的是，当CTT被移植到人类Cx26上形成嵌合蛋白Cx26-CTT时，这将消除人类Cx26对二氧化碳的敏感性。Cx26-CTT亚基有可能成为一种完美的遗传工具，具有从Cx26半通道移除二氧化碳敏感性的精致选择性，但所有其他功能，最重要的是缝隙连接的二氧化碳敏感性，保持不变。我们的项目试图记录Cx26-CTT与野生型(正常)Cx26的共组装，并表征Cx26-CTT与Cx26亚基在已完成的半通道中消除其二氧化碳敏感性所需的相对比例--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Cx26同源物的CTT来优化CTT的效力，以产生共同序列(CCTT)和最小序列(MCTT)，我们可以连接多个CTT以获得更大的效力。在这项开发工作的最后，我们将在体外表征Cx26-CTT在内源表达的Cx26中消除二氧化碳敏感性的有效性及其在半管和缝隙连接之间以及其他相关连接蛋白之间的选择性。在开发出一种有效和选择性的体外工具后，我们将在体内展示这项工作可以改变呼吸的敏感性。这将通过设计病毒来实现，这种病毒可以导致Cx26-CTT在脑干非常特定的细胞中表达，我们知道Cx26在调节呼吸方面起到了作用。我们的项目将开发并验证一套基因工具，它将实现前所未有的成就：选择性地从Cx26半脑中去除对二氧化碳的敏感性。这将是其他研究的强大推动力--例如，研究Cx26半通道如何有助于：控制整个生命过程中的呼吸；控制大脑中的血液流动，以及为什么这种控制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，Cx26对二氧化碳的敏感性如何有助于其他器官系统的生理。

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.