CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

Abstract

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2.DOI: http://dx.doi.org/10.7554/eLife.01213.001

Conditional depletion of methyl-CpG-binding protein 2 in astrocytes depresses the hypercapnic ventilatory response in mice

“…Opening of connexin 26 hemichannels in response to CO 2 is also an important mechanism for the release of ATP (28,37). The connexins (Cx43, Cx40, Cx45, Cx32) have been found to be expressed at normal levels in MeCP2-deficient mice (57).…”

CO2 carbamylation of proteins as a mechanism in physiology

“…Mutational studies confirmed this, with both of these residues being required for CO 2 -dependent channel opening in Cx26. Elastic network modelling studies confirmed that an electrostatic interaction between lysine 125 and arginine 104 in the neighbouring subunit was capable of causing channel opening in Cx26 by restricting movement of the individual subunits of the hexamer [22].…”

“…Sequence comparison of the CO 2 sensitive connexins (26,30 and 32) to the non-sensitive Cx31 indicated one lysine residue, lysine 125, that was conserved only in the CO 2 sensitive connexins [22] (Figure 2). This residue was found to be 6.5 Å (1 Å = 0.1 nm ) from arginine 104 on the neighbouring subunit and therefore capable of electrostatic interactions [22,23]. It was hypothesized that stabilization of the carbamate bond through a salt bridge to this positive arginine could replace the role usually filled by Mg 2 + ions.…”

Ammonia Mediates Cortical Hemichannel Dysfunction in Rodent Models of Chronic Liver Disease

“…Hypoxic conditions (without CBF) were next applied to unload the slice and also to demonstrate bidirectional permeability of the channels to CBF. (26) The same hypoxic stimulus was then reapplied in the presence of CBF, resulting in dye loading. After application of each stimulus in the presence of CBF, a further 5-minute perfusion with aCSF containing CBF was performed, followed by a 10-minute wash with normal aCSF, enabling the channels to return to their physiological state.…”

“…Hypoxic conditions, known to increase both lactate production due to inhibition of oxidative phosphorylation and the opening probability of connexin hemichannels, (13,24) were induced for 2-4 minutes by replacement of oxygen in the medium with nitrogen (perfusion of the chamber with aCSF saturated with 95% N 2 /5% CO 2 ). (25,26) Because detection of lactate by the biosensors requires oxygen (Supporting Fig. S1B), (27) the effect of hypoxia was determined by measuring the peak lactate release upon reoxygenation ( Fig.…”