New tools for investigating connexin26 hemichannel function in physiological systems

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.

We propose to develop genetic tools to selectively remove CO2-sensitivity from Cx26 hemichannels, while leaving that of Cx26 gap junctions unchanged. To do this, we shall exploit insights gained from studying the molecular evolution of Cx26: lungfish and amphibian Cx26 homologues have an extended C-terminal tail (CTT) that interferes with the CO2-dependent opening of the hemichannel, but does not affect CO2-dependent closing of the gap junction. When grafted onto human Cx26 to produce Cx26-CTT, the CTT prevents the hemichannels from opening to CO2.

We shall optimize Cx26-CTT to achieve a potent dominant negative effect, so that less than 3 Cx26-CTT subunits need assemble with WT Cx26 to remove CO2 sensitivity from the hexameric hemichannels. We shall document the coassembly of Cx26 WT and Cx26-CTT via FRET and the ability of Cx26-CTT to remove CO2 sensitivity from the hemichannel while leaving the gap junction unchanged. We shall also document the selectivity of Cx26-CTT for Cx26 versus Cx30 and Cx32 two closely related beta connexins that Cx26 can coassemble with. To allow in vivo investigations we shall devise a control construct that does not remove CO2 sensitivity from Cx26 WT to act as a comparator for Cx26-CTT.

We shall use the Cx26-CTT and control constructs to probe the CO2-dependent control of breathing in mice. We shall transduce cells in the ventral caudal medulla oblongata with viral vectors carrying these constructs to target the endogenous WT Cx26 at this site. We shall document the sensitivity of breathing to CO2 in the Cx26-CTT and control mice thereby gaining definitive evidence for the contribution of Cx26 hemichannels. We shall also use ATP biosensors to test whether Cx26-CTT attenuates CO2-dependent ATP release.

Our project will generate new genetic tools for the selective removal of CO2 sensitivity from Cx26 hemichannels, which will enable new investigations of this key property of Cx26 in a wide range of physiological contexts.

Beneficiaries:
Pharmaceutical sector
Clinicians, such as neonatologists and paediatricians
Schools, Deafness Charities
Cx26 patients and their carers

How will they benefit:
i) From our development of new genetic tools to investigate the CO2 sensitivity of Cx26 hemichannels and its role in cell to cell signalling.

ii) From the new knowledge that Cx26 hemichannels are critical to the chemosensory control of breathing -this sheds new light on how frequently-occurring mutations in Cx26 could affect respiratory drive, with consequences for the regulation of breathing during sleep.

Pharmaceutical sector (long term impact):
Our work will identify Cx26 hemichannels as an important target for therapies. These might include allosteric modulators to alter Cx26 gating or even restore CO2 sensitivity in mutant Cx26 -as the CO2-bindjng motif remains in place. Such drugs might restore breathing control in affected individuals, and might reduce or delay late onset hearing loss. Other interventions could utilize drugs that stimulate breathing by mechanisms independent of Cx26, thereby at least partially counteracting the effects of the mutations.

Clinicians (short to medium term impact):
Around 3:1000 people are affected by congenital hearing loss, 40% of which arises from nonsyndromic recessive mutations. Mutations in Cx26 are the commonest genetic cause of hearing loss. There are also much rarer syndromic missense dominant mutations in Cx26 that cause hearing loss and multiple health problems. We have shown that certain mutations, and combinations of mutations, will diminish the CO2-sensitivity of Cx26 and, for one of these mutations, can lead, in neonates, to frequent episodes of central sleep apnea. The outcomes of our project, plus further research enabled by these outcomes, will persuade clinicians to use detailed genotyping information from patients affected by syndromic or nonsyndromic hearing loss, to recognize a priori any potential for central sleep apnea. This in turn would inform a management strategy to ameliorate any consequent health issues and improve their quality of life. Central sleep apnea can trigger serious conditions such as heart disease and hypertension, and there is a clear benefit to the patient and health care systems (through reduced costs) from early recognition of this problem.

Deafness charities, schools, patients and their carers (short to medium term impact):
There is a need for an advocacy role by charities, schools, carers to persuade clinicians to take the potential risks of these mutations seriously, so that they can be better managed. We can empower this advocacy role by:

- Giving them precise knowledge of the key mutations in Cx26 that could affect CO2 sensitivity and respiratory drive.

- Disseminating information about risks of central sleep apnea and its potential consequences to patients and carers.

- Raising awareness of the potential for central apnea in teachers, parents and how this may adversely affect pupils' learning and attainment of developmental targets.