Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a ϳ6.5°rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca 2؉ -induced closure.Gap junction channels (GJC) 3 are dynamic macromolecular complexes capable of opening and closing the channel pore in response to a number of stimuli such as divalent cations, signaling molecules, phosphorylation, pH, and modulators of specific isoforms (1). These regulated conduits for the passage of small molecules greatly influence homeostasis, development, ionic transmission, and other cellular processes. Whereas there exist strong cell biological, biochemical, and biophysical evidence for the effects of these modulators, there is not much information at the structural level as to the conformational changes that occur in closing the pore in response to these stimuli.Each connexin (Cx) channel is composed of two hexamers (connexons) that dock at their apposed extracellular surfaces. The cyclic arrangement of the subunits within the hexamers suggests that gating can occur by a rotation and translation of the transmembrane segments within all six monomers. It has been postulated that gating occurs as a "camera iris" shutter (2). An alternate hypothesis has been proposed in which intra-connexin associations occur to produce either a particle-receptor blockage at the cytoplasmic surface (3, 4) or as a physical gate near the extracellular surface ("loop gate") (5). Whether these proposed mechanisms correlate to the closure of fast and/or slow gates that have been characterized by electrophysiological methods (see Ref. 6) remain to be determined.Gating by intracellular acidification is one way that connexin channels open and close in response to stimuli. Experimentally determined decreases in intracellular pH are known to decrease junctional electrical coupling in cardiomyocytes and in Purkinje fibers (7-10) as well as in teleost and amphibian embryos (11). Stergiopoulos et al. (12) showed that many, but not all, connexins close in a pH-sensit...