Structural and biophysical basis of Connexin26 channel mediated disease

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

Connexin26 (Cx26) forms both hemichannels and gap junctions, which are important for intercellular communication. We established in the brainstem that CO2 binds to and opens Cx26 hemichannels, allowing them to release ATP, which then activates the neural circuits that control breathing. Surprisingly, we have now found that CO2 closes Cx26 gap junctions. This closing action on gap junctions may be mediated by CO2-binding at the same motif that causes the hemichannel opening.

Many different Cx26 mutations are linked to human pathologies that range from hearing loss to syndromes that involve serious disorders of skin, vision and hearing. Unexpectedly, some of these Cx26 mutations also alter the CO2-sensitivity of Cx26 hemichannels. As both Cx26 gap junctions and hemichannels co-exist in tissues affected by Cx26 mutations, understanding the differential modulation of gap junctions and hemichannels by CO2 is fundamentally important.

We shall therefore analyze whether CO2 does indeed bind to the same site on Cx26 gap junctions and hemichannels. We shall mutate the key amino acids that comprise the CO2-binding site in hemichannels, and test whether this also alters the CO2-sensitivity of the gap junction. We shall test whether pathology-causing mutations of Cx26, which alter the sensitivity of hemichannels to CO2, also change the sensitivity of the gap junction to CO2.

To demonstrate at an atomic level how CO2 binds to Cx26 and opens the hemichannel, we shall use X-ray crystallographic methods on Cx26 (with and without CO2 bound). Human mutations that alter the CO2 sensitivity of Cx26 do not directly affect the CO2 binding site. Therefore we shall crystallize Cx26 mutants, with and without CO2 bound, to see how the structure has been altered and whether this can explain the altered CO2 sensitivity. We shall utilize a complementary method, cryoEM, to examine the conformational changes that follow CO2 binding.

Planned Impact

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

How will they benefit:

Clinicians (immediate to medium term impact):
Around 1:1000 people are affected by congenital hearing loss, about 50% of which arises from mutations in Cx26. Most cases of Cx26 hearing loss are non-syndromic, however there are also much more rare missense dominant mutations in Cx26 that cause syndromic hearing loss and multiple health problems. Our work will help to identify the mutations, and combinations of mutations, that affect the CO2-sensitivity of Cx26.

We have recently shown that loss of CO2-sensitivity, resulting from a dominant syndromic mutation in Cx26, caused frequent episodes of central sleep apnea in the affected individual. Non-syndromic recessive mutations that reduce CO2-sensitivity are also likely to affect breathing (in homozygotes or in combination with other recessive mutations). Clinicians, by utilizing detailed genotyping information in patients affected by syndromic or nonsyndromic hearing loss, would be able to recognize a priori any potential for central sleep apnea.

Apart from the obvious risk of death, central sleep apnea can trigger serious conditions such as heart disease and hypertension. There is thus a clear benefit to the patient and health care systems (through reduced costs) from early recognition of this problem and better management to ameliorate any consequent health issues.

Deafness charities (immediate to medium term impact):
Dissemination of information about risks of central sleep apnea and potential consequences to patients and carers. Development of guidance on potential management strategies for affected individuals. Definition of new research priorities around sleep apnea.

Schools (medium term impact):
Awareness of central apnea in affected pupils, and consequences for learning and attaining developmental targets. Prioritization of special needs assistance to those children most affected. Promoting knowledge and awareness to help children, parents and teachers to manage the condition.

Cx26 patients and their carers (medium term impact):
Precise knowledge of the key mutations in Cx26 that could affect CO2 sensitivity and respiratory drive would empower patients and their carers. Central sleep apnea is very difficult for affected individuals to recognise -it is likely to be manifest as tiredness. Lack of adequate sleep affects all aspects and stages of life. For affected children disrupted sleep may affect attainment at school through effects on ability to concentrate and disruption of memory consolidation. These children are likely to be suffering from some degree of hearing loss (and in the rare syndromic cases other health problems), which increases problems of communication with relevant clinicians and carers.

Defining more precisely patients at risk (from knowledge of mutations in Cx26) would increase awareness of these problems and allow management to minimize the negative effects on health and well-being. Any improved management of their health is likely to help them achieve key developmental and educational milestones and thus improve quality of life for their whole lifespan.

Pharmaceutical and biotech sector (long term impact):
Identification of strategies for therapeutic intervention: these could be drugs that specifically interact with Cx26 to restore CO2 sensitivity -the structural information that this project will provide will be invaluable in generating a rational drug design program. Such drugs might rescue cochlear development and prevent or reduce 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.
 
Description Direct evidence for carbamylation 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided purified Cx26, and expertise in conditions required to cause carbamylation of the native protein
Collaborator Contribution Our partners are using their novel MS methods to demonstrate carbamylation of lysine residues in Cx26
Impact Multidisciplinary: Mass Spectrometry, Protein purification, Structural biology, Physiology
Start Year 2019
 
Description Cafe Scientifique 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave a lecture on the sensory control of breathing and how a key molecule involved evolved some 400 MYA, and how mutations in this molecule impact on human health.
Year(s) Of Engagement Activity 2018
URL http://www.cafescientifique.org/index.php?option=com_contentbuilder&title=uk-leamington-spa&controll...
 
Description Cafe Scientifique 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I presented my work on the structural biology of Cx26 and implications for the evolution of breathing from fish to mammals
Year(s) Of Engagement Activity 2018
URL https://www.leamingtoncafesci.org/past-programme-all-to-be-formatted/
 
Description Public Science evening on Exoplanets and life elsewhere in the Universe 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The evening started with the physics of exoplanets, then considered the evolution of life (RNA hypothesis), then went on to consider what extremophiles on Earth can tell us about the potential for life on other bodies in the solar system and on exoplanets. After an interval the idea of a habitable base was subjected to serious scrutiny by a plant scientist, and I finished with a short presentation on the likelihood of finding a nervous system elsewhere in the Universe. This started with why we have nervous systems, how they evolved on Earth and the physics-chemical contraints that are imposed on nervous systems. My conclusion was that nervous systems elsewhere in the universe are quite likely (they are useful and have evolved on Earth twice), that they are likely to be constrained by the same physicochemical limits but are most probably very different in mechanistic detail.
Year(s) Of Engagement Activity 2019
 
Description Talk at Charles River 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact I gave a lecture on chemosensory processes -CO2 sensing in the brain stem and nutrient sensing in the hypothalamus. Around 40 researchers from the CRO Charles River attended
Year(s) Of Engagement Activity 2017
 
Description University of East Anglia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact I gave a research seminar on CO2 sensing by Cx26 -going from structural biology to physiology
Year(s) Of Engagement Activity 2018
 
Description Whatever have glial cells done for us? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave a talk on modern understanding of glial cell signalling -how they neuron-glia signalling occurs, and some of the functions that glial cells have such as the control of sleep, the chemosensory control of breathing and the role of specialised glial cells in the control of food intake
Year(s) Of Engagement Activity 2021
URL https://www.leamingtoncafesci.org/