New tools for investigating connexin26 hemichannel function in physiological systems

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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.