The extracellular surface of the gap junction cell-to-cell channels was imaged in phosphate-buffered saline with an atomic force microscope. The fully hydrated isolated gap junction membranes adsorbed to mica were irregular sheets approximately 1–2 microns across and 13.2 (+/- 1.3) nm thick. The top bilayer of the gap junction was dissected by increasing the force applied to the tip or sometimes by increasing the scan rate at moderate forces. The exposed extracellular surface revealed a hexagonal array with a center-to-center spacing of 9.4 (+/- 0.9) nm between individual channels (connexons). Images of individual connexons with a lateral resolution of < 3.5 nm, and in the best case approximately 2.5 nm, were reliably and reproducibly obtained with high-quality tips. These membrane channels protruded 1.4 (+/- 0.4) nm from the extracellular surface of the lipid membrane, and the atomic force microscope tip reached up to 0.7 nm into the pore, which opened up to a diameter of 3.8 (+/- 0.6) nm on the extracellular side.
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