Imaging Single-Cell Ca2+ Dynamics of Brainstem Neurons and Glia in Freely Behaving Mice.

In vivo brain imaging, using a combination of genetically encoded Ca2+ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum-a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond. Key features • We developed a protocol that allows imaging from brainstem neurons and glia in freely behaving rodents. • Our refined method of GRIN lenses implantation and cell sorting approach gives the highest number of cells with the least postoperative complications. • The revised deep brainstem imaging method paves way to understand complex behaviours such as cardiorespiratory regulation, sleep, swallowing, and digestion. • Our protocol can be implemented to image cerebellar structures to understand their role in key functions such as balance, movement, motor learning, and more.

Layer-Dependent Interaction Effects in the Electronic Structure of Twisted Bilayer Graphene Devices.

Near the magic angle, strong correlations drive many intriguing phases in twisted bilayer graphene (tBG) including unconventional superconductivity and chern insulation. Whether correlations can tune symmetry breaking phases in tBG at intermediate (≳ 2°) twist angles remains an open fundamental question. Here, using ARPES, we study the effects of many-body interactions and displacement field on the band structure of tBG devices at an intermediate (3°) twist angle. We observe a layer- and doping-dependent renormalization of bands at the K points that is qualitatively consistent with moiré models of the Hartree-Fock interaction. We provide evidence of correlation-enhanced inversion symmetry-breaking, manifested by gaps at the Dirac points that are tunable with doping. These results suggest that electronic interactions play a significant role in the physics of tBG even at intermediate twist angles and present a new pathway toward engineering band structure and symmetry-breaking phases in moiré heterostructures.

Channel-mediated ATP release in the nervous system.

ATP is well established as a transmitter and modulator in the peripheral and central nervous system. While conventional exocytotic release of ATP at synapses occurs, this transmitter is unusual in also being released into the extracellular space via large-pored plasma membrane channels. This review considers the channels that are known to be permeable to ATP and some of the functions of channel-mediated ATP release. While the possibility of ATP release via channels mediating volume transmission has been known for some time, localised ATP release via channels at specialised synapses made by taste cells to the afferent nerve has recently been documented in taste buds. This raises the prospect that "channel synapses" may occur in other contexts. However, volume transmission and channel synapses are not necessarily mutually exclusive. We suggest that certain glial cells in the brain stem and hypothalamus, which possess long processes and are known to release ATP, may be candidates for both modes of ATP release -channel-mediated volume transmission in the region of their somata and more localised transmission possibly via either conventional or channel synapses from their processes at distal targets. Finally, we consider the different characteristics of vesicular and channel synapses and suggest that channel synapses may be advantageous in requiring less energy than their conventional vesicular counterparts. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

Scapular Stress Fracture of the Inferior Angle in an Adolescent Swimmer.

We present the case of a competitive swimmer who was 14 years, 9 months old and had a 4-month history of posterior shoulder pain. She was initially evaluated by her school's trainer and completed a 2-week rehabilitation program, but pain returned with return to swimming. After feeling a "pop" while swimming with an increase in associated shoulder pain, the patient presented for medical evaluation. Plain radiographs were read as unremarkable. A formal physical therapy program resulted in increased pain, and the patient returned to clinic within 2 weeks with pain out of proportion to examination. Magnetic resonance imaging at this time identified a stress fracture along the inferior angle of the scapula, prompting a complete shutdown of activity for 4 weeks with vitamin D and calcium supplementation. A physical therapy regimen was restarted at 6 weeks with complete resolution of symptoms and return to swimming at 3 months. This case report is important because it highlights a sports-related stress fracture of the inferior angle of the scapula, a finding not currently present in the literature, in a swimmer, a sport not yet associated with scapular stress fractures. [Orthopedics. 2023;46(4):e249-e252.].

Neural correlate of reduced respiratory chemosensitivity during chronic epilepsy.

While central autonomic, cardiac, and/or respiratory dysfunction underlies sudden unexpected death in epilepsy (SUDEP), the specific neural mechanisms that lead to SUDEP remain to be determined. In this study, we took advantage of single-cell neuronal Ca2+ imaging and intrahippocampal kainic acid (KA)-induced chronic epilepsy in mice to investigate progressive changes in key cardiorespiratory brainstem circuits during chronic epilepsy. Weeks after induction of status epilepticus (SE), when mice were experiencing recurrent spontaneous seizures (chronic epilepsy), we observed that the adaptive ventilatory responses to hypercapnia were reduced for 5 weeks after SE induction with its partial recovery at week 7. These changes were paralleled by alterations in the chemosensory responses of neurons in the retrotrapezoid nucleus (RTN). Neurons that displayed adapting responses to hypercapnia were less prevalent and exhibited smaller responses over weeks 3-5, whereas neurons that displayed graded responses to hypercapnia became more prevalent by week 7. Over the same period, chemosensory responses of the presympathetic rostral ventrolateral medullary (RVLM) neurons showed no change. Mice with chronic epilepsy showed enhanced sensitivity to seizures, which invade the RTN and possibly put the chemosensory circuits at further risk of impairment. Our findings establish a dysfunctional breathing phenotype with its RTN neuronal correlate in mice with chronic epilepsy and suggest that the assessment of respiratory chemosensitivity may have the potential for identifying people at risk of SUDEP.

X-linked Charcot Marie Tooth mutations alter CO2 sensitivity of connexin32 hemichannels.

Connexin32 (Cx32) is expressed in myelinating Schwann cells. It forms both reflexive gap junctions, to facilitate transfer of molecules from the outer to the inner myelin layers and hemichannels at the paranode to permit action potential-evoked release of ATP into the extracellular space. Loss of function mutations in Cx32 cause X-linked Charcot Marie Tooth disease (CMTX), a slowly developing peripheral neuropathy. The mechanistic links between Cx32 mutations and CMTX are not well understood. As Cx32 hemichannels can be opened by increases in PCO2, we have examined whether CMTX mutations alter this CO2 sensitivity. By using Ca2+ imaging, dye loading and genetically encoded ATP sensors to measure ATP release, we have found 5 CMTX mutations that abolish the CO2 sensitivity of Cx32 hemichannels (A88D, 111-116 Del, C179Y, E102G, V139M). Others cause a partial loss (L56F, R220Stop, and R15W). Some CMTX mutations have no apparent effect on CO2 sensitivity (R15Q, L9F, G12S, V13L, V84I, W133R). The mutation R15W alters multiple additional aspects of hemichannel function including Ca2+ and ATP permeability. The mutations that abolish CO2 sensitivity are transdominant and abolish CO2 sensitivity of co-expressed Cx32WT. We have shown that Schwannoma RT4 D6P2T cells can release ATP in response to elevated PCO2 via the opening of Cx32. This is consistent with the hypothesis that the CO2 sensitivity of Cx32 may be important for maintenance of healthy myelin. Our data, showing a transdominant effect of certain CMTX mutations on CO2 sensitivity, may need to be taken into account in any future gene therapies for this condition.

Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice.

Regulation of systemic PCO2 is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO2 chemosensory nuclei mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pFL). Here, we use GCaMP6 expression and head-mounted mini-microscopes to image Ca2+ activity in these nuclei in awake adult mice during hypercapnia. Activity in the pFL supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO2, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours.

Revealing the order parameter dynamics of 1T-TiSe[Formula: see text] following optical excitation.

The formation of a charge density wave state is characterized by an order parameter. The way it is established provides unique information on both the role that correlation plays in driving the charge density wave formation and the mechanism behind its formation. Here we use time and angle resolved photoelectron spectroscopy to optically perturb the charge-density phase in 1T-TiSe[Formula: see text] and follow the recovery of its order parameter as a function of energy, momentum and excitation density. Our results reveal that two distinct orders contribute to the gap formation, a CDW order and pseudogap-like order, manifested by an overall robustness to optical excitation. A detailed analysis of the magnitude of the the gap as a function of excitation density and delay time reveals the excitonic long-range nature of the CDW gap and the short-range Jahn-Teller character of the pseudogap order. In contrast to the gap, the intensity of the folded Se[Formula: see text]* band can only give access to the excitonic order. These results provide new information into the the long standing debate on the origin of the gap in TiSe[Formula: see text] and place it in the same context of other quantum materials where a pseudogap phase appears to be a precursor of long-range order.

Conformational changes and CO2-induced channel gating in connexin26.

Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO2) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO2. We show a correlation between the level of PCO2 and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO2 alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.

Brain H+ /CO2 sensing and control by glial cells.

Maintenance of constant brain pH is critically important to support the activity of individual neurons, effective communication within the neuronal circuits, and, thus, efficient processing of information by the brain. This review article focuses on how glial cells detect and respond to changes in brain tissue pH and concentration of CO2 , and then trigger systemic and local adaptive mechanisms that ensure a stable milieu for the operation of brain circuits. We give a detailed account of the cellular and molecular mechanisms underlying sensitivity of glial cells to H+ and CO2 and discuss the role of glial chemosensitivity and signaling in operation of three key mechanisms that work in concert to keep the brain pH constant. We discuss evidence suggesting that astrocytes and marginal glial cells of the brainstem are critically important for central respiratory CO2 chemoreception-a fundamental physiological mechanism that regulates breathing in accord with changes in blood and brain pH and partial pressure of CO2 in order to maintain systemic pH homeostasis. We review evidence suggesting that astrocytes are also responsible for the maintenance of local brain tissue extracellular pH in conditions of variable acid loads associated with changes in the neuronal activity and metabolism, and discuss potential role of these glial cells in mediating the effects of CO2 on cerebral vasculature.

Novel Point-of-Care Diagnostic Method for Neonatal Encephalopathy Using Purine Nucleosides.

Background: Evidence suggests that earlier diagnosis and initiation of treatment immediately after birth is critical for improved neurodevelopmental outcomes following neonatal encephalopathy (NE). Current diagnostic tests are, however, mainly restricted to clinical diagnosis with no molecular tests available. Purines including adenosine are released during brain injury such as hypoxia and are also present in biofluids. Whether blood purine changes can be used to diagnose NE has not been investigated to date. Methods: Blood purines were measured in a mouse model of neonatal hypoxia and infants with NE using a novel point-of-care diagnostic technology (SMARTChip) based on the summated electrochemical detection of adenosine and adenosine metabolites in the blood. Results: Blood purine concentrations were ∼2-3-fold elevated following hypoxia in mice [2.77 ± 0.48 µM (Control) vs. 7.57 ± 1.41 µM (post-hypoxia), p = 0.029]. Data in infants with NE had a 2-3-fold elevation when compared to healthy controls [1.63 ± 0.47 µM (Control, N = 5) vs. 4.87 ± 0.92 µM (NE, N = 21), p = 0.0155]. ROC curve analysis demonstrates a high sensitivity (81%) and specificity (80%) for our approach to identify infants with NE. Moreover, blood purine concentrations were higher in infants with NE and seizures [8.13 ± 3.23 µM (with seizures, N = 5) vs. 3.86 ± 0.56 µM (without seizures, N = 16), p = 0.044]. Conclusion: Our data provides the proof-of-concept that measurement of blood purine concentrations via SMARTChip technology may offer a low-volume bedside test to support a rapid diagnosis of NE.

CO2-Sensitive Connexin Hemichannels in Neurons and Glia: Three Different Modes of Signalling?

Connexins can assemble into either gap junctions (between two cells) or hemichannels (from one cell to the extracellular space) and mediate cell-to-cell signalling. A subset of connexins (Cx26, Cx30, Cx32) are directly sensitive to CO2 and fluctuations in the level within a physiological range affect their open probability, and thus, change cell conductance. These connexins are primarily found on astrocytes or oligodendrocytes, where increased CO2 leads to ATP release, which acts on P2X and P2Y receptors of neighbouring neurons and changes excitability. CO2-sensitive hemichannels are also found on developing cortical neurons, where they play a role in producing spontaneous neuronal activity. It is plausible that the transient opening of hemichannels allows cation influx, leading to depolarisation. Recently, we have shown that dopaminergic neurons in the substantia nigra and GABAergic neurons in the VTA also express Cx26 hemichannels. An increase in the level of CO2 results in hemichannel opening, increasing whole-cell conductance, and decreasing neuronal excitability. We found that the expression of Cx26 in the dopaminergic neurons in the substantia nigra at P7-10 is transferred to glial cells by P17-21, displaying a shift from being inhibitory (to neuronal activity) in young mice, to potentially excitatory (via ATP release). Thus, Cx26 hemichannels could have three modes of signalling (release of ATP, excitatory flickering open and shut and inhibitory shunting) depending on where they are expressed (neurons or glia) and the stage of development.

Purines for Rapid Identification of Stroke Mimics (PRISM): study protocol for a diagnostic accuracy study.

BACKGROUND: Rapid treatment of stroke improves outcomes, but accurate early recognition can be challenging. Between 20 and 40% of patients suspected to have stroke by ambulance and emergency department staff later receive a non-stroke 'mimic' diagnosis after stroke specialist investigation. This early diagnostic uncertainty results in displacement of mimic patients from more appropriate services, inappropriate demands on stroke specialist resources and delayed access to specialist therapies for stroke patients. Blood purine concentrations rise rapidly during hypoxic tissue injury, which is a key mechanism of damage during acute stroke but is not typical in mimic conditions. A portable point of care fingerprick test has been developed to measure blood purine concentration which could be used to triage patients experiencing suspected stroke symptoms into those likely to have a non-stroke mimic condition and those likely to have true stroke. This study is evaluating test performance for identification of stroke mimic conditions. METHODS: Design: prospective observational cohort study Setting: regional UK ambulance and acute stroke services Participants: a convenience series of two populations will be tested: adults with a label of suspected stroke assigned (and tested) by attending ambulance personnel and adults with a label of suspected stroke assigned at hospital (who have not been tested by ambulance staff). INDEX TEST: SMARTChip Purine assay Reference standard tests: expert clinician opinion informed by brain imaging and/or other investigations will assign the following diagnoses which constitute the suspected stroke population: ischaemic stroke, haemorrhagic stroke, TIA and stroke mimic conditions. SAMPLE SIZE: ambulance population (powered for mimic sensitivity) 935 participants; hospital population (powered for mimic specificity) 377 participants. ANALYSES: area under the receiver operating curve (ROC) and optimal sensitivity, specificity, and negative and positive predictive values for identification of mimic conditions. Optimal threshold for the ambulance population will maximise sensitivity, minimum 80%, and aim to keep specificity above 70%. Optimal threshold for the hospital population will maximise specificity, minimum 80%, and aim to keep sensitivity above 70%. DISCUSSION: The results from this study will determine how accurately the SMARTChip purine assay test can identify stroke mimic conditions within the suspected stroke population. If acceptable performance is confirmed, deployment of the test in ambulances or emergency departments could enable more appropriate direction of patients to stroke or non-stroke services. TRIAL REGISTRATION: Registered with ISRCTN (identifier: ISRCTN22323981) on 13/02/2019 http://www.isrctn.com/ISRCTN22323981.

CO2 sensing by connexin26 and its role in the control of breathing.

Breathing is essential to provide the O2 required for metabolism and to remove its inevitable CO2 by-product. The rate and depth of breathing is controlled to regulate the excretion of CO2 to maintain the pH of arterial blood at physiological values. A widespread consensus is that chemosensory cells in the carotid body and brainstem measure blood and tissue pH and adjust the rate of breathing to ensure its homeostatic regulation. In this review, I shall consider the evidence that underlies this consensus and highlight historical data indicating that direct sensing of CO2 also plays a significant role in the regulation of breathing. I shall then review work from my laboratory that provides a molecular mechanism for the direct detection of CO2 via the gap junction protein connexin26 (Cx26) and demonstrates the contribution of this mechanism to the chemosensory regulation of breathing. As there are many pathological mutations of Cx26 in humans, I shall discuss which of these alter the CO2 sensitivity of Cx26 and the extent to which these mutations could affect human breathing. I finish by discussing the evolution of the CO2 sensitivity of Cx26 and its link to the evolution of amniotes.

Elevated blood purine levels as a biomarker of seizures and epilepsy.

OBJECTIVE: There is a major unmet need for a molecular biomarker of seizures or epilepsy that lends itself to fast, affordable detection in an easy-to-use point-of-care device. Purines such as adenosine triphosphate and adenosine are potent neuromodulators released during excessive neuronal activity that are also present in biofluids. Their biomarker potential for seizures and epilepsy in peripheral blood has, however, not yet been investigated. The aim of the present study was to determine whether blood purine nucleoside measurements can serve as a biomarker for the recent occurrence of seizures and to support the diagnosis of epilepsy. METHODS: Blood purine concentrations were measured via a point-of-care diagnostic technology based on the summated electrochemical detection of adenosine and adenosine breakdown products (inosine, hypoxanthine, and xanthine; SMARTChip). Measurements of blood purine concentrations were carried out using samples from mice subjected to intra-amygdala kainic acid-induced status epilepticus and in video-electroencephalogram (EEG)-monitored adult patients with epilepsy. RESULTS: In mice, blood purine concentrations were rapidly increased approximately two- to threefold after status epilepticus (2.32 ± .40 µmol·L-1 [control] vs. 8.93 ± 1.03 µmol·L-1 [after status epilepticus]), and levels correlated with seizure burden and postseizure neurodegeneration in the hippocampus. Blood purine concentrations were also elevated in patients with video-EEG-diagnosed epilepsy (2.39 ± .34 µmol·L-1 [control, n = 13] vs. 4.35 ± .38 µmol·L-1 [epilepsy, n = 26]). SIGNIFICANCE: Our data provide proof of concept that the measurement of blood purine concentrations may offer a rapid, low-volume bedside test to support the diagnosis of seizures and epilepsy.

Magnetic Resonance Imaging Findings After Superior Capsule Reconstruction.

PURPOSE: To evaluate graft properties on magnetic resonance imaging (MRI) after superior capsular reconstruction (SCR) to help improve our understanding of postoperative imaging. METHODS: We identified consecutive patients who underwent SCR by a single surgeon and who had postoperative MRIs available. MRIs were analyzed to look for common postoperative findings on imaging. RESULTS: Ten consecutive patients with an average age of 58 years who underwent SCR by a single surgeon had postoperative MRIs on average 404 days from surgery. Eight patients had completely intact grafts on follow-up MRI. All intact grafts were similar with homogenous appearance on all coronal images. All patients displayed some trace fluid with mild heterogenous signal at the level of the glenoid, which could represent failure of the graft to completely incorporate at the level of the glenoid or could be normal in the postoperative setting since all eight intact grafts displayed this finding. None of the patients with intact grafts had bony edema noted on either the glenoid or humeral side. Four of 8 patients were noted to have trace bony edema at the level of the lateral acromion. One patient had complete disruption on the glenoid side. One patient had partially intact graft that revealed heterogenous appearance of graft. CONCLUSIONS: An intact graft displays a more homogenous signal on consecutive postoperative MRI coronal images than disrupted grafts or partially intact grafts. This suggests that intact grafts have better clinical outcomes than a partially disrupted or completely disrupted graft. However, the finding of heterogenous signal/fluid at the glenoid graft interface in all intact grafts could not be explained in this study. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Biological insights from the direct measurement of purine release.

Although purinergic signalling has been a well-established and accepted mechanism of chemical communication for many years, it remains important to measure the extracellular concentration of ATP and adenosine in real time. In this review I summarize the reasons why such measurements are still needed, how they provide additional mechanistic insight and give an overview of the techniques currently available to make spatially localised measurements of ATP and adenosine in real time. To illustrate the impact of direct real-time measurements, I explore CO2 and nutrient sensing in the medulla oblongata and hypothalamus. In both of these examples, the sensing involves hemichannel mediated ATP release from glial cells. For CO2 the hemichannels involved, connexin26, are directly CO2-sensitive. This mechanism contributes to the chemosensory control of breathing. In the hypothamalus, specialised glial cells, tanycytes, directly contact the cerebrospinal fluid in the 3rd ventricle and sense nutrients via sweet and umami taste receptors. Nutrient sensing by tanycytes is likely to contribute to the control of body weight as their selective stimulation alters food intake. To illustrate the importance of direct adenosine measurements, I consider the complex and multiple mechanisms of activity-dependent adenosine release in different brain regions. This activity dependent release of adenosine is likely to mediate important feedback regulation and may also be involved in controlling the sleep-wake state. I finish by briefly considering the potential of whole blood purine measurements in clinical practice.

Real-time measurement of adenosine and ATP release in the central nervous system.

This brief review recounts how, stimulated by the work of Geoff Burnstock, I developed biosensors that allowed direct real-time measurement of ATP and adenosine during neural function. The initial impetus to create an adenosine biosensor came from trying to understand how ATP and adenosine-modulated motor pattern generation in the frog embryo spinal cord. Early biosensor measurements demonstrated slow accumulation of adenosine during motor activity. Subsequent application of these biosensors characterized real-time release of adenosine in in vitro models of brain ischaemia, and this line of work has recently led to clinical measurements of whole blood purine levels in patients undergoing carotid artery surgery or stroke. In parallel, the wish to understand the role of ATP signalling in the chemosensory regulation of breathing stimulated the development of ATP biosensors. This revealed that release of ATP from the chemosensory areas of the medulla oblongata preceded adaptive changes in breathing, triggered adaptive changes in breathing via activation of P2 receptors, and ultimately led to the discovery of connexin26 as a channel that mediates CO2-gated release of ATP from cells.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

KEY POINTS: A moderate increase in PCO2 (55 mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. ABSTRACT: Cx26 hemichannels open in response to moderate elevations of CO2 ( PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30 mm propionate, we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.