Structural and biophysical basis of Connexin26 channel mediated disease

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CO2 is the unavoidable by-product of metabolism and its concentration controls the acidity of blood. Because only a small increase in the acidity of blood can prove fatal, the regulated excretion of CO2 via breathing is an extremely important life-preserving process. We discovered that CO2 binds to and opens membrane channels formed from Connexin26 (Cx26), allowing them to release ATP, which then activates the neural circuits that control breathing. This is a key mechanism for the CO2-dependent regulation of breathing.Cx26 is one of 20 human connexin genes. It encodes a membrane channel that can dock to identical membrane channels in adjacent cells, to form a "gap junction". Gap junctions allow direct passage of ions and small molecules between cells. In addition, undocked connexin membrane channels, "hemichannels", can permit release of signalling substances such as the neurotransmitter ATP. Both gap junctions and hemichannels provide important but distinctive mechanisms for cell-to-cell communication.Cx26 is critical for human physiology -over 100 different Cx26 mutations have been linked to human pathologies. Cx26 mutations are the commonest genetic cause of hearing loss. Other Cx26 mutations cause potentially fatal syndromes that involve serious disorders of skin, vision and hearing. Unexpectedly, some of the Cx26 mutations that cause hearing loss and syndromes also alter the CO2-sensitivity of Cx26 hemichannels. CO2-dependent signalling via Cx26 may therefore have further vital, yet currently unrecognised, roles in human physiology. Surprisingly, we have now found that CO2 closes Cx26 gap junctions in contrast to its opening action on hemichannels. This closing action of CO2 on gap junctions may occur as a result of binding to the same location in the protein that causes the opening of the hemichannel. This is extremely important, as both Cx26 gap junctions and hemichannels co-exist in the same tissues, such as those involved in the control of breathing and hearing. Understanding the differential modulation of gap junctions and hemichannels by CO2 is thus fundamentally important and will provide new insight into the aetiology of pathologies linked to mutations of Cx26.We shall analyze whether CO2 does indeed bind to the same site on gap junctions and hemichannels, by mutating the key amino acids that comprise the CO2-binding site in hemichannels to test whether this also alters the CO2-sensitivity of the gap junction. We shall then test whether the pathology-causing mutations of Cx26, which alter the sensitivity of hemichannels to CO2, also change the sensitivity of the gap junction to CO2.To understand exactly how CO2 binds to Cx26 and opens the hemichannel, we need atomic level structures of the Cx26 in various states. We shall purify Cx26, grow crystals (with and without CO2 bound) and use X-ray methods to determine the atomic structures. As the human mutations that alter the CO2 sensitivity of Cx26 do not affect the CO2 binding site, it is unclear why they should have this effect. Therefore we shall crystallize mutant variants of Cx26, with and without CO2 bound, to see how the structure has been altered and whether this can explain the altered CO2 sensitivity. We shall also explore whether a complementary method, which does not require protein crystals, can provide structural information at sufficient resolution.Our research will show how CO2 binds to Cx26 and how the channels open and close. This will provide the structural underpinnings to one of the most important life preserving reflexes -the CO2-dependent regulation of breathing. Additionally, we will transform mechanistic understanding of how certain Cx26 mutations linked to human pathology alter CO2 binding. This may suggest therapies to lessen pathology, and management strategies to enhance patients' quality of life. This new structural information may aid development of drugs to rescue the CO2-sensitivity of the mutated Cx26 protein.