FIGURE 2.

Purified GAAPs and hBI-1 exhibit ion channel activity in planar lipid bilayers. A and B, biochemical analyses of purified CMLV GAAP, VACV Evans GAAP, and hBI-1. A, UV absorbance profile of purified vGAAPs and hBI-1 during SEC. *, protein aggregation peak. Fractions corresponding to monomeric and oligomeric populations of vGAAPs and hBI-1 were pooled (A, bracket) and concentrated, and their contents were analyzed by non-reducing SDS-PAGE and Coomassie staining (B). The expected positions of the monomeric (×1) and oligomeric proteins (×2, ×3, and ×4) are shown. C, bilayer chamber used. A planar lipid bilayer is formed across a micrometer-sized aperture within the chip. GUVs reconstituted with purified protein are added to the cis chamber (ground), allowing the incorporation of protein into the bilayer. The KCl concentration is greater in the trans relative to the cis chamber. D, electrophysiological recordings from artificial lipid bilayers reconstituted with purified hBI-1, VACV Evans GAAP, or CMLV GAAP show spontaneous channel openings. Representative current traces were recorded at the indicated holding potentials, which are expressed as the potential on the cis side relative to the trans side. Downward deflections of the current trace represent positive ions flowing from the trans to the cis side of the bilayer. The lipid bilayer alone (n = 35), after the addition of GUVs reconstituted in the presence of lauryldimethylamine N-oxide (n = 10), or reconstituted with A_2AR (n = 6) was used as a negative control. Dotted line, closed state.