Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43

Abstract

Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.Gap junctions are aqueous channels that connect neighboring cells electrically and chemically by mediating permeation of inorganic ions and solutes of M r Ͻ 1000 (Ref. 1 and reviewed in Ref.2). They are formed by two connexons or gap-junctional hemichannels in series with each connexon conformed by six connexin molecules (3-5). In addition, gap-junctional hemichannels have been identified in the plasma membrane of native cells and cell lines (6 -8) where they appear to participate in a number of physiological and pathophysiological processes (6 -15). Because of the roles of gap-junctional channels in development, organ function, and disease processes and the possible physiological and pathophysiological roles of gap-junctional hemichannels (see Ref. 2), there is considerable interest in understanding the structure and regulation of connexons. Despite all the effort devoted to study connexins, basic aspects of the structure and function of the protein remain unknown.The connexin 43 isoform (Cx43) 1 is expressed in organs such as the brain, myocardium, and kidney as well as in capillary endothelial cells (16 -22). A medium resolution structure of this connexin, without the C-terminal domain, has been solved by cryoelectron microscopy of two-dimensional crystals (4, 5). These structural studies, however, have not provided unambiguous information on helix packing, the residues that form the pore, or the structure of the regulatory C-terminal domain. Studies in which native residues were substituted with Cys and then reacted with thiol reagents, however, suggest that transmembran...

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

“…On the other hand, the Cx43 Δ245 mutant is not able to confer responsiveness to bisphosphonates or to PTH (Plotkin et al ., 2002; Bivi et al ., 2011). Cx43‐silenced cells expressing the Cx43 C‐tail alone or Cx43 Cys‐less , a mutant that due to the lack of cysteine residues in the extracellular domain (Dahl et al ., 1991) only forms hemichannels and it is unable for form gap junctions (Bao et al ., 2004; Tong et al ., 2007), maintained an elevated percentage of apoptosis, similar to vector‐transfected cells. This contrasts with the reversal of the effect of Cx43 silencing in osteoblastic cells by the same Cx43 Cys‐less mutant (Bivi et al ., 2011).…”

Reversible Pore Block of Connexin Channels by Cyclodextrins

“…Therefore, the reconstituted channels are oriented cytoplasmic side outward. Intriguingly, this corresponds to the native topology one would expect for connexin channels in Golgi/endoplasmic reticulum prior to plasma membrane insertion, and to that of in vitro translated connexin inserted co-translationally (Cx32 and (57). Because CDs act only from the extraliposomal side, these studies show that they interact with connexin pores only from the ''intracellular'' aspect of the channel and do not have any detectable (by TSF) inhibitory effects on connexin channel permeability from the ''extracellular'' side.…”

“…Molecular permeability of the reconstituted channels was assessed by TSF of the liposomes. TSF has been well characterized (42,43,51) and effectively used in studies of connexin channel permeability and modulation (25,39,40,(52)(53)(54)(55)(56)(57). TSF separates liposomes into distinct populations within a density gradient on the basis of the permeability of an open connexin channel to two solutes of different densities (typically urea and sucrose; see "Experimental Procedures").…”

Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

“…Phosphorylation state may be relevant, and metabolic inhibition induces dephosphorylation of Cx43 hemichannels, as indicated by increased electrophoretic mobility. ¶ However, dephosphorylated Cx43 hemichannels remain open in a reconstituted system (25) and when expressed in Xenopus laevis oocytes (26). Trolox and DTT do not prevent dephosphorylation of Cx43 hemichannels during metabolic inhibition (10,24).…”