How the brain senses CO2

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences

Abstract

Metabolism requires O2 and produces CO2 as a by-product. The amount of dissolved CO2 controls the acidity (pH) of our body fluids including blood. Many physiological processes are sensitive to pH, thus efficient control of the amount of dissolved CO2 in blood is a critical homoeostatic function. Special mechanisms exist to measure the amount of dissolved CO2 in blood ?if there is too much we breathe faster to drive off the excess, if there is too little we breathe less frequently. While there is evidence that CO2 is detected indirectly as pH, we have discovered a new molecular mechanism for the direct detection of CO2 ?CO2 binds to a type of channel called connexin 26 (Cx26), and in doing so causes it to open and release a chemical called ATP that activates neurons. Cx26 is sensitive to CO2 in exactly the physiological range required ?our normal levels of dissolved CO2 are at the mid-point of channel activation. Cx26 can thus respond to both increases and decreases in dissolved CO2. Cx26 is present in the correct areas of the brain to help control breathing. Our programme seeks to develop the genetic tools to analyze with great rigour the contributions of Cx26 to the measurement of CO2 and the control of breathing. Use of these tools will not only enable us to establish the causal link between Cx26 and behaviour, but also to determine the tissues, regions and cell types that are important for the detection of CO2. We also wish to understand the mechanism by which CO2 binds to the channel to change its conformation and cause it to open. We shall mix and match portions of CO2-sensitive and non-CO2-sensitive connexins to endow a previously non-sensitive connexin with CO2 sensitivity. This will tell us which part of the protein is important. We shall then identify the precise amino acid involved by mutating single amino acids in the critical region of the molecule. In parallel with this we shall use an analytical technique called NMR spectroscopy to directly and definitively test one possible way that CO2 could bind to the protein. Our proposal has the potential to transform understanding of how the brain senses CO2. This is likely to be important in understanding how CO2-sensing may be altered during pathologies such as congestive heart failure and chronic obstructive pulmonary disease.

Technical Summary

We seek to understand the mechanisms by which brain senses the partial pressure of CO2 (PCO2) in arterial blood. Chemosensory reflexes regulate breathing to keep arterial PCO2 (and PO2) within tightly defined limits and thus maintain acid-base balance. Although disorders of chemosensory mechanisms are potentially life threatening, especially in the context of other pathologies such as congestive heart failure, knowledge of their underlying mechanisms remains surprisingly incomplete. We have recently discovered a new molecular transducer for CO2 chemoreception ?the connexin 26 (Cx26) hemichannel. During elevated arterial PCO2 (hypercapnia), increased CO2 binding to Cx26 causes it to open more and hence release more ATP. This ATP excites the respiratory network and causes the adaptive increase in breathing to hypercapnia. Cx26 is also sensitive to decreases in PCO2; closure of the channel, resulting in reduced ATP release, decreases the respiratory drive during hypocapnia and consequently helps to maintain PCO2. Cx26 is widely distributed throughout the brain in the subpial astrocytes and leptomeninges. This raises two important and major questions. Which are the key tissues and cell types for respiratory chemosensing ?the astrocytes or the leptomeninges? How can gating of a gap junction hemichannel be directly altered by CO2? We shall address these two problems in a coordinated fashion. Firstly, we shall develop the transgenic tools to delete Cx26 in selected cell types and regions. We shall then examine the effect on responses to CO2. This will provide definitive causal evidence linking Cx26 to the behavioural adaptive reflexes in responses to changes in PCO2 and will allow us to determine the most critical cellular components and regions for central CO2 chemosensing. Secondly, we shall make chimeric constructs by inserting structural motifs of Cx26 (CO2-sensitive) into a non-CO2-sensitive homologous connexin to identify the critical domains of the molecule. We shall complement the identification of key structural components by loss-of-function mutagenesis. We shall definitively test our favoured hypothesis that CO2 alters Cx26 hemichannel gating via carbamylation of a lysine residue by utilizing 15N-13C NMR spectroscopy to demonstrate directly the existence of the hypothesized carbamate group. The NMR studies will assist the design of the mutagenesis strategy and complement the functional evaluation of the mutant channels: we expect the existence of a carbamate group to be present in those mutant channels that are CO2-sensitive and absent in those that are not.
 
Description Project Grant
Amount £173,000 (GBP)
Funding ID RPG-2015-090 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 12/2016
 
Title Cx26 Mutants 
Description Various mutations of Cx26 and related connexins to probe mechanism of CO2 binding 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This has allowed us to understand the nature of binding of CO2 to Cx26 and these mutations are central to the published eLife paper. We have now found a mutation of Cx26 which is linked to major human pathology which removes the CO2 sensitivity of Cx26 
 
Title Dominant negative Cx26 
Description This is a dominant negative subunit of Cx26 which is designed to remove the CO2 sensitivity of wild type Cx26 subunits. We have demonstrated this with cultured cells and are designing viral constructs to express it in a cell specific manner, to delete CO2 sensing in a very targeted fashion. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact None yet 
 
Title Targetted deletion of Cx26 
Description generation of cre-lox crosses that selectively delete Cx26 in particular cell populations in medulla of mice -neural crest-deriveed, GFAP-expressing and leptomeningeal 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The deletion from neural-crest-dervied cells in medulla oblongata and GFAP-expressing cells in medulla oblongata gives a substantially reduced sensitivity to CO2 
 
Description Cx26 molecular gating 
Organisation Durham University
Department School of Biological and Biomedical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the idea and intellectual background -specifically our prediction of which amino acid residues CO2 binds to in Cx26
Collaborator Contribution The partners ran elastic network models of Cx26 gating motions with and without CO2 bound to the predicted site which gave insight into the mechanism of CO2 action
Impact Meigh, L, 2013, eLife Multidisciplinary: bioinformatics; site directed mutagenesis; elastic network modelling; patch clamp; image analysis
Start Year 2012
 
Description Cx26, CO2 sensing and human disease 
Organisation University of Connecticut
Country United States 
Sector Academic/University 
PI Contribution We evaluated the CO2 sensitivity of the Cx26 mutant p.Ala88Val and found that this mutation linked to keratitis ichthyosis deafness (KID) syndrome completely removed the CO2 sensitivity of Cx26. We have also shown that this mutation acts in a dominant manner to remove CO2 sensitivity.
Collaborator Contribution They provided the case study of the infant with KID syndrome, and cardiorespiratory recordings demonstrating that this infant suffered from reduced respiratory drive, and the identification of the mutation in Cx26
Impact Meigh, L., Hussain, N., Mulkey, D.K. and Dale, N. (2014) Connexin26 hemichannels with a mutation that causes KID syndrome in humans lack sensitivity to CO2. eLife 10.7554/eLife.04249.
Start Year 2014
 
Description Aging brain 
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 A Public Science Evening entitled "Is aging inevitable?" I chaired the evening and participated in special session on brain ageing. As well as considering cognitive decline with age and dementia, we considered critical physiological functions (breathing) controlled by small populations of neurons that have to last an entire lifetime, and the possibilities of adult neural stem cells
Year(s) Of Engagement Activity 2018
 
Description Brain Night 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Part of Brain Awareness Week 2013. Helped to arrange and run a Brain Night at the Virgins and Castles Pub, Kenilworth. This consisted of a quiz, and presentation of various materials from the Dana foundation, and informal conversations with members of the public about the brain and our research.

None
Year(s) Of Engagement Activity 2013
 
Description Brain anatomy -golgi staining of neurons and synapses 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Performed three times, make parents and school children aware of the beauty of neurons and importance of synaptic spines and how these change with aging. Interactive discussion about Alzheimer's disease, Parkinson's and stroke.

This has encouraged students to come to Warwick to study Neuroscience.
Year(s) Of Engagement Activity 2011,2012,2013
 
Description Brain event 
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 Display of brain neuroanatomy, and information. Small group conversations with members of public about aspects of brain function and implications for society: e.g. how memories are stored; control of breathing; control of body weight through regulating food intake; healthy raging.
Year(s) Of Engagement Activity 2017
URL http://www.theherbert.org/whats_on/104/amazing_brain
 
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 Evolution of Cx26 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Press release on paper in Proc Roy Soc B on "Evolutionary adaptation of the sensitivity of connexin26 hemichannels to CO2". Picked up by 11 news outlets and 2 bloggers.
Year(s) Of Engagement Activity 2017
URL https://www.altmetric.com/details/16020978
 
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/