Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC

Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-0437, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2007; 104(12):4919-24. DOI: 10.1073/pnas.0603154104
Source: PubMed


Gap-junctional channels, permeable to large hydrophilic solutes of up to M(r) approximately 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits. The subunit composition was determined by luminescence resonance energy transfer. We show that all Cx43 subunits in the hemichannel hexamer have to be phosphorylated to abolish sucrose (M(r) 342) permeability. We also show that the hemichannel pores with all subunits phosphorylated by PKC have a sizable diameter, allowing for permeation of the small hydrophilic solute ethyleneglycol (M(r) 62). These results indicate that phosphorylation of Cx43 by PKC alters the hemichannel size selectivity and explain why PKC activity affects dye transfer between cells without consistent effects on electrical communication.

Download full-text


Available from: Sung Lee, Dec 27, 2013
10 Reads
  • Source
    • "The channel protein CX43 plays a role in the apoptotic process by regulating the permeability of small molecules, including adenosine 5′-triphosphate, adenosine diphosphate, adenosine, cAMP, inositol-1,4,5-triphosphate, glutamate, and glutathione [14], [15], [40]–[42]. In response to hypoxia, AGS8 organizes the complex that includes Gβγ, CX43, and possibly kinases that initiate phosphorylation of CX43 [13]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Signaling via heterotrimeric G-protein is involved in the development of human diseases including ischemia-reperfusion injury of the heart. We previously identified an ischemia-inducible G-protein activator, activator of G-protein signaling 8 (AGS8), which regulates Gβγ signaling and plays a key role in the hypoxia-induced apoptosis of cardiomyocytes. Here, we attempted to intervene in the AGS8-Gβγ signaling process and protect cardiomyocytes from hypoxia-induced apoptosis with a peptide that disrupted the AGS8-Gβγ interaction. Synthesized AGS8-peptides, with amino acid sequences based on those of the Gβγ-binding domain of AGS8, successfully inhibited the association of AGS8 with Gβγ. The AGS8-peptide effectively blocked hypoxia-induced apoptosis of cardiomyocytes, as determined by DNA end-labeling and an increase in cleaved caspase-3. AGS8-peptide also inhibited the change in localization/permeability of channel protein connexin 43, which was mediated by AGS8-Gβγ under hypoxia. Small compounds that inhibit a wide range of Gβγ signals caused deleterious effects in cardiomyocytes. In contrast, AGS8-peptide did not cause cell damage under normoxia, suggesting an advantage inherent in targeted disruption of the AGS8-Gβγ signaling pathway. These data indicate a pivotal role for the interaction of AGS8 with Gβγ in hypoxia-induced apoptosis of cardiomyocytes, and suggest that targeted disruption of the AGS8-Gβγ signal provides a novel approach for protecting the myocardium against ischemic injury.
    PLoS ONE 03/2014; 9(3):e91980. DOI:10.1371/journal.pone.0091980 · 3.23 Impact Factor
  • Source
    • "Fortunately, Cx43 HC of controlled subunit composition can be generated in vitro from purified Cx43. As mentioned under “Charaterization of purified connexin hemichannels,” purified Cx43 (but not Cx26) solubilized in detergent forms HCs where the connexins (subunits) exchange (Bao et al., 2007). This allowed us to generate and reconstitute Cx43 HCs of controlled average subunit composition. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gap-junction channels (GJCs) are aqueous channels that communicate adjacent cells. They are formed by head-to-head association of two hemichannels (HCs), one from each of the adjacent cells. Functional HCs are connexin hexamers composed of one or more connexin isoforms. Deafness is the most frequent sensineural disorder, and mutations of Cx26 are the most common cause of genetic deafness. Cx43 is the most ubiquitous connexin, expressed in many organs, tissues, and cell types, including heart, brain, and kidney. Alterations in its expression and function play important roles in the pathophysiology of very frequent medical problems such as those related to cardiac and brain ischemia. There is extensive information on the relationship between phosphorylation and Cx43 targeting, location, and function from experiments in cells and organs in normal and pathological conditions. However, the molecular mechanisms of Cx43 regulation by phosphorylation are hard to tackle in complex systems. Here, we present the use of purified HCs as a model for functional and structural studies. Cx26 and Cx43 are the only isoforms that have been purified, reconstituted, and subjected to functional and structural analysis. Purified Cx26 and Cx43 HCs have properties compatible with those demonstrated in cells, and present methodologies for the functional analysis of purified HCs reconstituted in liposomes. We show that phosphorylation of serine 368 by PKC produces a partial closure of the Cx43 HCs, changing solute selectivity. We also present evidence that the effect of phosphorylation is highly cooperative, requiring modification of several connexin subunits, and that phosphorylation of serine 368 elicits conformational changes in the purified HCs. The use of purified HCs is starting to provide critical data to understand the regulation of HCs at the molecular level.
    Frontiers in Physiology 02/2014; 5:71. DOI:10.3389/fphys.2014.00071 · 3.53 Impact Factor
  • Source
    • "Interestingly, a progressive dephosphorylation of this protein was also observed in parallel with the metabolic inhibition progression. But even when Cx43 is dephosphorylated, which is a signaling to induce hemichannel opening (Bao et al., 2007), hemichannels were closed by Trolox indicating that the redox status of Cx43 is a control mechanism more important than dephosphorylation , at least in metabolic stress conditions. Then, Retamal et al. (2006) studied the molecular mechanism that control Cx43 hemichannel opening under metabolic inhibition. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Connexins (Cxs) and Pannexins (Panxs) are two non-related protein families, having both the property to form hemichannels at the plasma membrane. There are 21 genes coding for different Cx based proteins and only 3 for Panx. Under physiological conditions, these hemichannels (Cxs and Panxs) present a low open probability, but when open, they allow the release of signaling molecules to the extracellular space. However, under pathological conditions, these hemichannels increase their open probability, inducing important lysis of metabolites, and ionic imbalance, which in turn induce the massive entry of Ca(+2) to the cell. Actually, it is well recognized that Cxs and Panxs based channels play an important role in several diseases and -in many cases- this is associated with an aberrant hemichannel opening. Hemichannel opening and closing are controlled by a plethora of signaling including changes of the voltage plasma membrane, protein-protein interactions, and several posttranslational modifications, including protein cleavage, phosphorylation, glycosylation, hydroxylation and S-nitrosylation, among others. In particular, it has been recently shown that the cellular redox status modulates the opening/closing and permeability of at least Cx43, Cx46, and Panx1 hemichannels. Thus, for example, the gaseous transmitter nitric oxide (NO) can induce the S-nitrosylation of these proteins modulating in turn several of their properties. The reason is that the redox status of a cell is fundamental to set their response to the environment and also plays an important role in several pathologies. In this review, I will discuss how NO and other molecules associated with redox signaling modulate Cxs and Panx hemichannels properties.
    Frontiers in Physiology 02/2014; 5:80. DOI:10.3389/fphys.2014.00080 · 3.53 Impact Factor
Show more