Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Abstract

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...

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

“…Prior to SMFS, membranes containing densely packed SteT were located by contact mode AFM imaging. If necessary, the AFM stylus was used as a nanoscalpel to remove aggregates or the upper layer of the vesicles (47,48). An unperturbed area of the membrane patch was selected, and the AFM stylus was pushed onto the membrane at a force of ϳ750 pN for 0.5-1.0 s to promote unspecific attachment of the SteT polypeptide to the AFM stylus.…”

Structure determination of channel and transport proteins by high-resolution microscopy techniques

“…Because biomolecules are 'alive' under these native conditions, AFM has been successfully used to observe single proteins at work; for reviews, see Engel et al (1999) and Engel and Müller (2000). One of the most exciting examples in the literature for the direct visualization of conformational changes is the pH and Ca 2q -dependent opening and closing of the gap junction channel connexin26 Yu et al, 2007). Because of these unique capabilities, AFM is currently being used as a complementary technique to XRC and electron microscopy.…”

Molecular imaging of membrane proteins and microfilaments using atomic force microscopy

“…KirVac3.1 is one member of a family of transmembrane potassium channels, and gating of Kir channels is spontaneous but modulated by intracellular molecules such as lipids (PIP2), G-proteins, nucleotides, and ions (H + , Ca 2+ , Mg 2+ ). Yu et al (2007) demonstrated that pH-induced conformational changes in connexin 26 hemichannels were modulated by aminosulfonate. They removed the upper layer of the gap junction channel by repetitive scanning AFM tip with loading of strong force.…”

“…Efforts to observe the ultra-structures of cells using AFM have gradually increased since its invention Muller and Engel, 1999;Kreplak et al, 2007;Jaroslawski et al, 2007;Yu et al, 2007). Early studies of cells with AFM were focused on topological observation by high-reso-lution imaging.…”

“…AFM has been used for the study of membrane channels because it allows observation of cell surfaces in their native environment at remarkable resolution (Jaroslawski et al 2007;Yu et al, 2007;Muller, 2008;Stewart et al, 2010). Jaroslawski et al (2007) have directly visualized gating of KirBac3.1 potassium channels by AFM imaging.…”