Fig. 5.

EM image analysis of negatively stained Panx1 oligomers. (A) Preparations purified from baculovirus-infected Sf9 cells as detected on a Sypro Ruby-stained protein gel (Gel Stain) and a corresponding Western Blot (WB). (B, C) Two representative low dose electron micrographs for uranyl acetate-stained purified Panx1 channels diluted in water (B, unstimulated condition) and in KCl 50 mM (C, stimulated condition). These micrographs were recorded with a standard low dose imaging protocol (47) to minimize electron beam damage and preserve higher resolution structural features. The scale bar represents 50 nm. The insets on the top of the micrographs are three-fold enlargements of the encircled particles. The rectangle indicates one channel with the cytoplasmic view of the Panx1 channel facing up, and the circle indicates a channel with the extracellular view facing up. (D) Representative class averages (Class Avg) and hexagonal-symmetrized averages (Hexagonal Avg) of cytoplasmic and extracellular surface views of unstimulated and stimulated Panx1 channels. Shown also are averages and standard deviations from 10 measurements of the channel and pore diameters of 4–5 class averages for both views from the two conditions. (E) Scatter plot of the measurements shown in (D, n=10 for each measurement). +/−KCl Cyto Ptcl, stimulated and unstimulated cytoplasmic particle outer diameter; +/− KCl Extracell Ptcl, stimulated and unstimulated extracellular particle outer diameter