Conformational changes and CO2-induced channel gating in connexin26

Brotherton, Deborah; Savva, Christos G.; Ragan, Timothy J.; Dale, Nicholas; Cameron, Alexander D.

doi:10.1016/j.str.2022.02.010

Cited by 18 publications

(63 citation statements)

References 64 publications

(169 reference statements)

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“…With a functional Cx43 synthetic cell system, we turned our attention to suppressing Cx43 nanopore activity. Previous work in cells showed that Cx43 function is sensitive to cytoplasmic domain modification, 61 and recent Cryo-EM structures of connexon pores, 44,45,48 including Cx43's, 46,47 present an opportunity to use structure-guided design to engineer inhibition of Cx43. For Cx43, two solved structures show that the N-terminus provides key contacts at the bottom of the pore (cytoplasmic side) and faces inward toward the vacant channel (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Fortunately, several pore structures of connexins have recently been solved by Cryo-EM, opening the door for structureguided design of an activatable pore. [44][45][46][47][48] Here we report the light-activated assembly of a connexon nanopore in synthetic cells and the subsequent release of internal contents. We re-engineer connexin-43 (Cx43) 46,47 with a bulky protease recognition domain, which allows membrane-protein association but prevents nanopore function in the membrane.…”

Section: Introductionmentioning

confidence: 92%

“…Fortunately, several pore structures of connexins have recently been solved by Cryo-EM, opening the door for structure-guided design of an activatable pore. 44–48…”

Section: Introductionmentioning

confidence: 99%

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Light-activated assembly of connexon nanopores in synthetic cells

Sihorwala

Lin

Stachowiak

et al. 2022

Preprint

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During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Non-living systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One non-living system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexin nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

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Light-activated assembly of connexon nanopores in synthetic cells

Sihorwala

Lin

Stachowiak

et al. 2022

Preprint

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“…In contrast to acetyl and methyl small-molecule PTMs which are often catalyzed by acetylases and methyltransferases, respectively, amine carbamylation by CO 2 occurs independently of enzymatic activity and is readily reversible. Long-lived and physiologically relevant protein carbamates are thought to necessitate privileged binding sites with proximal cationic residues to stabilize the charged product, , such as the functional CO 2 binding sites of hemoglobin and RuBisCO . Temporary carbamylation events conversely occur on all surface amines to varying degrees and lifetimes which are largely dependent on the transition state energies and population of the unprotonated, reactive species.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific Detection and Characterization of Ubiquitin Carbamylation

et al. 2022

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The physiological consequences of varying in vivo CO2 levels point to a general mechanism for CO2 to influence cellular homeostasis beyond regulating pH. Aside from a few instances where CO2 has been observed to cause post-translational protein modification, by forming long-lived carbamates, little is known about how transitory and ubiquitous carbamylation events could induce a physiological response. Ubiquitin is a versatile protein involved in a multitude of cellular signaling pathways as polymeric chains of various lengths formed through one of the seven lysines or N-terminal amine. Unique polyubiquitin (polyUb) compositions present recognition signals for specific ubiquitin-receptors which enables this one protein to be involved in many different cellular processes. Advances in proteomic methods have allowed the capture and identification of protein carbamates in vivo, and Ub was found carbamylated at lysines K48 and K33. This was shown to negatively regulate ubiquitin-mediated signaling by inhibiting polyUb chain formation. Here, we expand upon these observations by characterizing the carbamylation susceptibility for all Ub amines simultaneously. Using NMR methods which directly probe 15N resonances, we determined carbamylation rates under various environmental conditions and related them to the intrinsic pK as. Our results show that the relatively low pK as for half of the Ub amines are correlated with enhanced susceptibility to carbamylation under physiological conditions. Two of these carbamylated amines, not observed by chemical capture, appear to be physiologically relevant post-translational modifications. These findings point to a mechanism for varying the levels of CO2 due to intracellular localization, cellular stresses, and metabolism to affect certain polyUb-mediated signaling pathways.

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“…Carbamylation of K125 by CO 2 forms a carbamate bridge between subunits that leads to the opening of the channel; ( c ) cartoon representation of CO 2 binding (green circles) to K125 (blue) and formation of the carbamate bridge to R104 (red) leading to channel opening. While the exact nature of the conformational change leading to channel opening is unclear, it is hypothesized to involve movement of the N termini of the six subunits which individually are in a helical secondary conformation (Brotherton et al, 2020; Maeda et al, 2009)…”

Section: Sensing Co2mentioning

confidence: 99%

Brain H⁺/CO₂ sensing and control by glial cells

Gourine

Dale

2022

Glia

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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.

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Conformational changes and CO2-induced channel gating in connexin26

Cited by 18 publications

References 64 publications

Light-activated assembly of connexon nanopores in synthetic cells

Light-activated assembly of connexon nanopores in synthetic cells

Site-Specific Detection and Characterization of Ubiquitin Carbamylation

Brain H⁺/CO₂ sensing and control by glial cells

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Conformational changes and CO2-induced channel gating in connexin26

Cited by 18 publications

References 64 publications

Light-activated assembly of connexon nanopores in synthetic cells

Light-activated assembly of connexon nanopores in synthetic cells

Site-Specific Detection and Characterization of Ubiquitin Carbamylation

Brain H+/CO2 sensing and control by glial cells

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Brain H⁺/CO₂ sensing and control by glial cells