Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species
Highlights
► Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions. ► Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. ► The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. ► Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations.
Introduction
The coordination of numerous cellular processes requires complex electric and metabolic cell–cell interactions. In vertebrate cells, these interactions are in part mediated by low-resistance intercellular channels at gap junctions (GJs). In addition, cells release chemical signals that act in an autocrine or paracrine manner, and one mode of release is through connexin (Cx) “hemichannels” (HCs), the precursors of gap junction channels each of which is formed by two HCs, one from each apposed cell.
HCs are formed by the oligomerization of six connexin subunits, which are encoded by 21 different genes in humans [1]. HCs can be formed of a single Cx (homomeric) or multiple Cxs (heteromeric). Members of a recently described three-member protein (1–3) family unrelated to the Cxs, termed pannexins (Panxs), can also form HCs in the cell membrane of vertebrates. Frequently, these two HC types are co-expressed, and their roles in membrane permeability can be differentiated by several means including their differential sensitivity to shRNA, to pharmacological inhibitors, to mimetic peptides with sequences the same as the extracellular loops of the Cx or Panx1, to extracellular divalent cations (opening of Cx HCs but not Panx HCs is enhanced by divalent free extracellular saline solutions), and to trivalent cations (e.g., La3+), which block Cx HCs but not Panx HCs [2].
Cx HC-mediated cell permeabilization can be enhanced by both physiological and pathological stimuli [3]. Supporting their role in paracrine and/or autocrine cell signaling, Cx HCs have been shown to be permeable to various metabolites, including glucose, glutathione, ascorbate, and NAD+ [4], [5], [6], [7], which is in agreement with their proposed role as pathways for cellular nutrition, detoxification, and propagation of Ca2+ waves [3].
The functional expression of Cx proteins fused to enhanced green fluorescent protein (EGFP) has facilitated the electrophysiological characterization of Cx43 HCs [8], [9]; in only a few studies were qualitative permeability properties of homomeric HCs assessed [10], [11], [12] and quantitative kinetic properties of permeation of Cx HCs remain largely unknown. The aim of the present study was to determine the diffusion kinetics of three cations of different size, shape and charge through mouse Cx43-EGFP and Cx43-WT HCs.
Section snippets
Reagents
HEPES, LaCl3 and ethidium (Etd) bromide were from Sigma–Aldrich (St. Louis, MO, USA). Propidium (Prd) iodide and 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride were from Molecular Probes, Inc. (Eugene, OR, USA). Gap26 (VCYDKSFPISHVR, first extracellular loop domain of Cx43) and 10pnx1 (WRQAAFVDSY, first extracellular loop domain of Panx1) peptides were obtained from NeoMPS, SA. (Strasbourg, France).
Cell culture
Experiments were performed on previously described parental HeLa cells or on these cells
Uptake of cationic permeability tracers by Cx43-EGFP HeLa cells is cooperative at low tracer concentration
HeLa cells transfected with specific Cxs express HCs at the cell surface, through which they take up and release small molecules including fluorescent dyes commonly used to assay whether they are open [8]. However, in cultured cells under resting conditions the presence of physiological concentrations of extracellular divalent cations reduces the open probability of HCs [8], [14], [15], so that longer times are required to determine HC permeability. Since divalent cation-free solution (DCFS)
Discussion
Here, we show that Cx43-EGFP HCs in cells bathed with DCFS allow positive cooperative transport across the cell membrane for low concentrations of Etd, Prd and DAPI and saturation at higher concentrations. Moreover, Cx43-EGFP HCs have different numbers of affinity sites for each permeant as suggested by the difference in from the Hill coefficients, and at least Etd and DAPI compete for passage through the same channels.
This work provides the first description of kinetic properties of HC
Acknowledgments
This work was partially supported by CONICYT (24080055 to JAO), FONDECYT (1111033 to JCS), FONDEF (D07I1086 to JCS and KSC), NHI (NS55363 to MVL) and Anillo (ACT71 to JCS) grants.
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2019, Journal of Biological ChemistryCitation Excerpt :It has generally been inferred that a connexin in its hemichannel configuration acts as a “half gap junction” and thus displays a permeability profile akin to that. Nonbiological fluorescent dyes are routinely used as a permeability proxy for ions or biological molecules based on dye size and charge in both gap junction and hemichannel research (20, 25–27). As an emerging indicator for the selectivity of Cx43 in its hemichannel configuration, a growing number of research groups have detected Cx43-mediated dye uptake at negative membrane potentials, at which DCFS-induced Cx43 hemichannel conductance is undetectable in mammalian cells (cell lines and primary cultures) as well as in Xenopus oocytes (11, 29, 30, 45–48).
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