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

Abstract

Gap-junctional channels, permeable to large hydrophilic solutes of up to M r Ϸ 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.luminescence energy transfer ͉ membrane protein ͉ transport

Proteoliposome Engineering with Cell‐Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems

“…Post‐translational modification of the C‐terminal of Cx43 regulates the biological activity and structure of the hexameric Cx43 hemichannel. In particular, Cx43 phosphorylation abolished the gate permeability of the hemichannel pore 14. As we previously discussed,[[qv: 9c]] PURESYSTEM is composed only of transcription/translation related factors with no phosphatases and the synthesized proteins are not phosphorylated.…”

Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers into Monomers during the Hydrolysis Cycle

“…If random association between monomers occurs after the addition of ATP, the sensitized emission originates from a representative sample of the dimers; those formed by a Tb 3ϩ monomer and a fluorescein monomer. Because the recorded acceptor emission with long lifetime can only arise from energy transfer, the stoichiometry of labeling affects the intensity of the signal, but not the distance calculations or the K d determinations, which depend on relative changes and probe lifetimes, respectively (18,19). In the absence of ATP, basically all NBDs are in monomeric form (supplemental Fig.…”

“…LRET has been used previously to assess intersubunit and intramolecular distances in various proteins (19,(22)(23)(24)(25)(26). LRET has many advantages for our experiments when compared with traditional fluorescence resonance energy transfer (19). These advantages include a very low background, high signal-to-noise ratio, and independence of the labeling stoichiometry, enabling LRET to measure distances in the 25-100 Å range with little uncertainty due to orientation factors (19).…”

“…For LRET, we used the rare element Tb 3ϩ , characterized by a long lifetime emission, as donor (18). LRET has been used previously to assess intersubunit and intramolecular distances in various proteins (19,(22)(23)(24)(25)(26). LRET has many advantages for our experiments when compared with traditional fluorescence resonance energy transfer (19).…”

“…LRET has many advantages for our experiments when compared with traditional fluorescence resonance energy transfer (19). These advantages include a very low background, high signal-to-noise ratio, and independence of the labeling stoichiometry, enabling LRET to measure distances in the 25-100 Å range with little uncertainty due to orientation factors (19). The residue at position 14 (Cys-14) was chosen as the target for labeling because it is a surface residue exposed to the aqueous solvent (available for labeling), it is not part of the active site or dimer interface (Fig.…”