Permeation of Calcium through Purified Connexin 26 Hemichannels

Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

“…2A shows that the hydrodynamic properties of WT MsbA, MsbA CL, and T561C, evaluated by gel filtration chromatography, are essentially identical. The elution is consistent with the expected homodimer of ϳ134 kDa because it occurs in close proximity to that of mouse P-glycoprotein (ABCB1, ϳ140 kDa) and the connexin 26 (Cx26) hexamer (ϳ160 kDa), two proteins that we routinely use in our laboratory (27,28). The Fig.…”

“…Protein concentration was determined by the BCA assay (Bio-Rad) and from the absorbance at 280 nm; purity was estimated at Ͼ95% from the gel filtration chromatogram and SDS-PAGE gels stained with Coomassie Blue. Monodispersity was assayed by dynamic light scattering as described (28).…”

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

“…Third, while progress in reconstituting proteo-liposomes has been made for in vitro refolding and assembly of purified channel membrane proteins using various liposome technologies [7], it is not known whether such systems can indeed function for channel activity in an in vivo cell system. Fourth, there is increasing evidence that different compositions of lipids modulate channel activities, for example reconstituted connexins [8], and it is yet to be elucidated whether such assembled connexin proteins in liposomes accurately resemble their native hemi-channels [9]. Furthermore, because most membrane proteins, especially eukaryotic proteins, require special environments for folding, assembly, post-modification, and trafficking, the direct correlation of in vitro liposomes studies with their cellular physiological functions in cells has not been achieved.…”

Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels

“…This DNA, optimized for E. coli expression, was subcloned into the pQE60 plasmid, and expression was carried out in XL10-Gold cells grown in a modified M9 medium. The purification procedure was as previously described for Cx26, using a combination of metal affinity (Talon Co 2ϩ column, Talon Superflow, Clontech) and gel-filtration chromatography (Superdex 200HR 10/300 GL column, GE Healthcare) (23). The tag was removed by incubation with tobacco etch virus protease for 12 h, at a Cx46/tobacco etch virus protease ratio of 1:15 (w/w).…”