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• W2049654315 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 ∼6.5° rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure. 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 ∼6.5° rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure. Gap junction channels (GJC) 3The abbreviations used are: GJC, gap junction channels; AFM, atomic force microscopy; Cx26, connexin26; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; N, Newton; TSF, transport-specific fractionation method; FWHM, full width half-maximum height. are dynamic macromolecular complexes capable of opening and closing the channel pore in response to a number of stimuli such as divalent cations, signal-ing molecules, phosphorylation, pH, and modulators of specific isoforms (1Harris A.L. Quart. Rev. Biophys. 2001; 34: 325-472Crossref PubMed Google Scholar). 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 (2Unwin P.N.T. Ennis P.D. Nature. 1984; 307: 609-613Crossref PubMed Scopus (266) Google Scholar). An alternate hypothesis has been proposed in which intra-connexin associations occur to produce either a particle-receptor blockage at the cytoplasmic surface (3Delmar M. Stergiopoulos K. Homma N. Calero G. Morley G. Ek-Vitorin J.F. Taffet S.M. Peracchia C. Gap Junctions. Molecular Basis of Cell Communication in Health and Disease. Vol. 49. Academic Press, San Diego2000: 223-248Google Scholar, 4Ek-Vitorin J.F. Calero G. Morley G.E. Coombs W. Taffet S.M. Delmar M. Biophys. J. 1996; 71: 1273-1284Abstract Full Text PDF PubMed Scopus (144) Google Scholar) or as a physical gate near the extracellular surface (“loop gate”) (5Trexler E.B. Bennett M.V.L. Bargiello T.A. Verselis V.K. Proc. Natl. Acad. Sci. 1996; 93: 5836-5841Crossref PubMed Scopus (268) Google Scholar). Whether these proposed mechanisms correlate to the closure of fast and/or slow gates that have been characterized by electrophysiological methods (see Ref. 6Bukauskas F.F. Verselis V.K. Biochim. Biophys. Acta. 2004; 1662: 42-60Crossref PubMed Scopus (244) Google Scholar) 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 (7Spray D.C. Burt J.M. Am. J. Physiol. 1990; 258: C195-C205Crossref PubMed Google Scholar, 8Noma A. Tsuboi N. J. Physiol. 1987; 382: 193-211Crossref PubMed Scopus (236) Google Scholar, 9Reber W.R. Weingart R. J. Physiol. 1982; 328: 87-104Crossref PubMed Scopus (66) Google Scholar, 10Burt J.M. Am. J. Physiol. 1987; 253: C607-C612Crossref PubMed Google Scholar) as well as in teleost and amphibian embryos (11Spray D.C. Harris A.L. Bennett M.V.L. Science. 1981; 211: 712-715Crossref PubMed Scopus (342) Google Scholar). Stergiopoulos et al. (12Stergiopoulos K. Alvarado J.L. Mastroianni M. Ek-Vitorin J.F. Taffet S.M. Delmar M. Circ. Res. 1999; 84: 1144-1155Crossref PubMed Scopus (123) Google Scholar) showed that many, but not all, connexins close in a pH-sensitive manner when tested in the paired Xenopus oocyte system. For example, Cx26 channels are pH-sensitive, but Cx32 channels are much less sensitive. Homomeric hemichannels also displayed this pH regulation (13Francis D. Stergiopoulos K. Ek-Vitorin J.F. Cao F.L. Taffet S.M. Delmar M. Dev. Genet. 1999; 24: 123-136Crossref PubMed Scopus (69) Google Scholar). Differences in the pH regulation of gap junctions were attributable to the diversity of the primary sequence, particularly in certain regions such as the C-terminal tail because the pH sensitivity of the dodecamer channels could be modified only when they were composed of heterotypic combinations (13Francis D. Stergiopoulos K. Ek-Vitorin J.F. Cao F.L. Taffet S.M. Delmar M. Dev. Genet. 1999; 24: 123-136Crossref PubMed Scopus (69) Google Scholar). However, it is important to note that these experiments were done in whole cells and cannot distinguish between gating of the channel because of protonation of the connexin or protonation of modulators or ligands that bind to the connexin and then close the channel. Bevans et al. (14Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) used another assay system to test for functional pore size called the transport-specific fractionation method (TSF). Heteromeric Cx32/Cx26 connexons reconstituted into liposomes showed pH-dependent channel activity as measured by a permeability assay when suspended in aminosulfonate buffers (e.g. HEPES, TAPS, MES). One of the simplest of these aminosulfonate compounds is taurine, a naturally occurring ubiquitous cytoplasmic component (15Huxtable R.J. Physiol. Rev. 1992; 72: 101-163Crossref PubMed Scopus (2266) Google Scholar, 16Wright C.E. Tallan H.H. Lin Y.Y. Gaull G.E. Annu. Rev. Biochem. 1986; 55: 427-453Crossref PubMed Google Scholar). This pH sensitivity was directly attributed to binding of protonated aminosulfonates to Cx26, because homomeric Cx32 channels did not show this pH sensitivity (17Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18Tao L. Harris A.L. J. Biol. Chem. 2004; 279: 38544-38554Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, it should be noted that Cx46 hemichannels in excised patches appear to display pH sensitivity in the absence of any added cytosolic material (19Trexler E.B. Bukauskas F.F. Bennett M.V. Bargiello T.A. Verselis V.K. J. Gen. Physiol. 1999; 113: 721-742Crossref PubMed Scopus (118) Google Scholar). Previously, we had shown using atomic force microscopy (AFM) that force-dissected connexin26 (Cx26) gap junction hemichannels reversibly open and close in response to Ca2+ acting as ligand (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar). These conformational changes could be only observed on extracellular hemichannel surfaces because the cytoplasmic GJC surface appears too flexible to be imaged at sufficiently high resolution to assign structural changes. This flexibility of the cytoplasmic surface is well documented not only by AFM studies (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar, 21Hoh J. Sosinsky G.E. Revel J.-P. Hansma P.K. Biophys. J. 1993; 65: 149-163Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 22Liu F. Arce F.T. Ramachandran S. Lal R. J. Biol. Chem. 2006; 281: 23207-23217Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) but also by electron microscopy and by other structural and biochemical methods. (For a complete review of flexibility in the cytoplasmic domains see Ref. 23Sosinsky G.E. Nicholson B.J. Biochim. Biophys. Acta. 2005; 1711: 99-125Crossref PubMed Scopus (199) Google Scholar.) In addition to our previous work, we have been able to image the extracellular surface at higher spatial resolution, allowing insight into the tertiary conformations of polypeptide loops connecting the transmembrane α-helices lining the connexon pore. Remarkably, gating events are well visualized at the extracellular surface. Taking the advantage of AFM to work under freely adjustable physiological conditions, we directly observed the reaction of hemichannels to pH changes of the buffer solution. All experiments were performed at ambient temperatures with fully hydrated proteins under buffer conditions. This simplistic system allows for the in vitro correlation of adding modulating agents and the resulting conformational changes directly imaged with AFM. The high-resolution AFM topographs suggest that the extracellular connexin domains undergo an aminosulfonate-modulated conformational change that closes the connexon channel at the extracellular surface. This conformational change was fully reversible. From these high-resolution AFM topographs, we propose that the mechanism for closure of the extracellular “loop” gate is different from the Ca2+-induced closure observed previously. Cx26 Gap Junction Preparation—Gap junctions were purified from overexpressing stably transfected HeLa cells grown to confluency by following a previously published procedure (24Hand G.M. Muller D.J. Nicholson B.J. Engel A. Sosinsky G.E. J. Mol. Biol. 2002; 315: 587-600Crossref PubMed Scopus (38) Google Scholar) with one modification. For Cx26 gap junctions, HEPES buffer was used instead of Tris buffer because preparations contained more gap junctions and less contaminating material as judged by conventional negative stain transmission electron microscopy. AFM Imaging—AFM topographs were recorded in buffer solution using contact mode. The AFM (Nanoscope III, di-Veeco) was equipped with a fluid cell and oxide-sharpened Si3N4 cantilevers (OMCL TR400PSA, Olympus, Japan), which had a nominal spring constant of ≈0.09 N/m. Prior to imaging, gap junction membranes were adsorbed to freshly cleaved mica as described (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar, 25Muller D.J. Amrein M. Engel A. J. Struct. Biol. 1997; 119: 172-188Crossref PubMed Scopus (267) Google Scholar). High-resolution topographs were recorded at minimal contact forces of ≤50 pN, which were manually adjusted to compensate for thermal drift (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar, 26Muller D.J. Fotiadis D. Scheuring S. Muller S.A. Engel A. Biophys. J. 1999; 76: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Proportional and integral gains were adjusted manually to minimize the error (deflection) signal and to maximize the height signal (27Putman C.A.J. van der Werft K. de Grooth B.G. van Hulst N.F. Greve J. Hansma P.K. SPIE. 1992; 1639: 198-204Crossref Google Scholar). When approaching a lateral resolution of ≈1 nm the scanning speed of the AFM tip was between 500 and 1.500 nm/s. Only topographs showing identical structural features scanning the same sample area in trace and retrace direction were selected for further analysis. Topographs showing asymmetric particles or indicating any kind of tip artifacts were not analyzed. Image Processing and Averaging—Topographs (512 × 512 pixel) were selected by the structural details of the protein imaged reproducibly and by comparing the simultaneously monitored height profiles acquired in trace and retrace direction. Correlation averaging was performed using the SEMPER image processing system (28Saxton W.O. Pitt T.J. Horner M. Ultramicroscopy. 1979; 4: 343-354Crossref Scopus (330) Google Scholar). A well preserved unit cell was selected from the raw data and cross-correlated with the topograph (29Saxton W.O. Baumeister W. J. Micros. 1982; 127: 127-138Crossref PubMed Scopus (696) Google Scholar). Unit cells were extracted according to the peak coordinates of the cross-correlated topograph. Single particle averages were generated by translationally and rotationally aligning the unit cells to a reference connexon and then averaged. This correlation average was used as reference for refinement cycles (30Saxton W.O. Baumeister W. Hahn M. Ultramicroscopy. 1984; 13: 57-70Crossref PubMed Scopus (210) Google Scholar). Correlation-averaged unit cells were 6-fold symmetrized. To assess the standard deviation σk,l, individual unit cells were extracted according to the coordinates of their correlation peaks and were aligned angularly as well as translationally before single particle averaging (31Frank J. Bretaudiere J.P. Carazo J.M. Verschoor A. Wagenknecht T. J Microsc. 1988; 150: 99-115Crossref PubMed Scopus (68) Google Scholar). The S.D. was then calculated from the averaged topograph μk,l for each pixel (k, l) for xi particles (32Schabert F.A. Engel A. Biophys. J. 1994; 67: 2394-2403Abstract Full Text PDF PubMed Scopus (120) Google Scholar) as shown in Equation 1.σk,l2=1N∑i=1N(Xk,li-μk,l)2(Eq.1) These S.D. maps are displayed as an image in a one-to-one pixel correspondence with the correlation-averaged topograph. The values range from 0.1 (black) to 0.4 (white) nm with the color table continuously ranging from black to white. Previously, we had shown that high-resolution AFM topographs of the extracellular surface could be obtained reproducibly. We also imaged the cytoplasmic surface; but because of the enhanced flexibility of the cytoplasmic domains (23Sosinsky G.E. Nicholson B.J. Biochim. Biophys. Acta. 2005; 1711: 99-125Crossref PubMed Scopus (199) Google Scholar), topographs could be obtained only at very low resolution, which would not allow the revealing of delicate conformational changes such as those observed on the relatively rigid extracellular surface of the hemichannel (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar). High-Resolution Imaging of the Extracellular Connexon Surface—Force dissection with AFM imaging provides high-resolution surface views of the extracellular surface. Fig. 1A shows a gap junction plaque imaged by AFM in buffer solution. As reported previously, Cx26 gap junctions exhibited a thickness of 17.5 ± 0.8 nm (n = 20), while the surrounding lipid membranes were only 4.5 ± 0.6-nm high (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar). These gap junction plaques exposed their cytoplasmic surfaces to the AFM tip, while the extracellular surfaces were sandwiched between the membranes embedding the bridging connexons. To characterize the extracellular surface, the upper connexon layer of the plaque had to be mechanically removed by the scanning AFM tip (Fig. 1B). At minimal forces of ≤50 pN applied to the scanning AFM cantilever, the repetitive imaging is non-destructive and allows reproducible observation of substructures of proteins embedded in biological membranes in their native conformation (26Muller D.J. Fotiadis D. Scheuring S. Muller S.A. Engel A. Biophys. J. 1999; 76: 1101-1111Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). However, at about 10–20-fold increased forces, the scanning process of the AFM allowed mechanical removal of the upper layer of the gap junction plaque (20Muller D.J. Hand G.M. Engel A. Sosinsky G.E. EMBO J. 2002; 21: 3598-3607Crossref PubMed Scopus (212) Google Scholar, 33Hoh J.H. Lal R. John S.A. Revel J.-P. Arnsdorf M.F. Science. 1991; 235: 1405-1408Crossref Scopus (209) Google Scholar, 34Fotiadis D. Scheuring S. Muller S.A. Engel A. Muller D.J. Micron. 2002; 33: 385-397Crossref PubMed Scopus (354) Google Scholar). After removal of the upper layer, the exposed extracellular surface could be observed at high resolution (Fig. 1C). The unprocessed AFM topograph shows the 6-fold symmetry of each connexon with its characteristic central pore. Each subunit of the connexon is thought to be formed by one connexin showing substructural details (23Sosinsky G.E. Nicholson B.J. Biochim. Biophys. Acta. 2005; 1711: 99-125Crossref PubMed Scopus (199) Google Scholar). The correlation average of the connexon surface (Fig. 1, D and E) showed their common structural details, whereas the S.D. map (Fig. 1, F and G) marked regions exhibiting an enhanced structural flexibility. Observing pH-induced Conformational Changes—Visualization of pH-induced conformational changes of the extracellular connexon surface was first performed under constant electrolyte concentration (2 mm EGTA, 1 mm phenylmethylsulfonyl fluoride) and at a constant HEPES concentration of 10 mm (Fig. 2). Prior to imaging, the membranes were absorbed onto the mica support using buffers containing 10 mm HEPES, 2 mm EGTA, 200 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride. Connexons observed at pH 6.0 appear very different to those observed at pH 7.0 and higher. However, it could appear that single connexons show individual deviations among each other. For example, a small number of connexons (<5%) showed different channel diameters than others. To allow statistical relevant conclusions about the average structural conformation of a single connexon under a certain buffer condition, we calculated single particle averages (Fig. 2, G–J, top row). The unprocessed topographs of single connexons and their corresponding averages did not show any significant differences in connexon shape or substructure. The majority of single connexons imaged (>85%) exhibited the same structural conformation as reflected by their average. Thus, we can rule out that the connexons imaged may have represented a mixed state of several conformational states, but rather a single predominant configuration. Connexon averages were computed from AFM topographs recorded at pH 6.0 (Fig. 2G, top), 6.5 (Fig. 2H, top), 7.0 (Fig. 2I, top), and 7.6 (Fig. 2J, top). Averages calculated from connexons imaged at pH 8.0 and 8.5 did not show any significant deviation from that recorded at 7.6 and therefore are not shown. Regions of S.D. maps (Fig. 2, G–J, bottom row) exhibiting enhanced values indicate for structural fluctuations of the connexon surface. All averages show the 8° skew of the connexon lobes from the vertical axis characteristic of detergent-treated samples (35Baker T.S. Caspar D.L.D. Hollingshead C.J. Goodenough D.A. J. Cell Biol. 1983; 96: 204-216Crossref PubMed Scopus (44) Google Scholar). In these averages, the connexon is displayed with a left skew. Whereas this overall appearance of the hexameric connexon does not change, the diameter of the central pore significantly increased with increasing pH. Besides measuring the channel diameters of averaged connexons, we analyzed those from single connexons. The histograms (Fig. 3) suggest that the average channel diameter, measured for a certain pH value reflects that of the majority (>85%) of the individual connexons. Under experimental conditions known to induce a closed channel (12Stergiopoulos K. Alvarado J.L. Mastroianni M. Ek-Vitorin J.F. Taffet S.M. Delmar M. Circ. Res. 1999; 84: 1144-1155Crossref PubMed Scopus (123) Google Scholar, 17Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), the channel entrance exhibited a maximum depth of 0.4 ± 0.1 nm with a diameter of only 0.6 ± 0.2 nm measured at full width half-maximum height (FWHM). The S.D. map of the average (Fig. 2G, bottom) shows no maxima at the channel entrance, suggesting that this area exhibited no enhanced structural flexibility. The surface structure of the connexon did not significantly change after increasing the pH to 6.5. However, the S.D. map of the closed connexon changed. The region at the channel entrance showed a slightly enhanced value of 0.15 nm (Fig. 2H, bottom), indicating that the corresponding structures now provided some structural flexibility. This may also explain the slightly increased maximum depth of the channel (0.5 ± 0.3 nm). At this pH, the width of the channel entrance increased slightly to 0.9 ± 0.3 nm (n = 91). Further increasing the pH to 7.0 further increased the width of the channel entrance (Fig. 2I, top) to 1.3 ± 0.2 nm (n = 85). As a result of this opening, the AFM tip now could penetrate into the channel entrance, detecting an average maximum depth of 1.2 ± 0.35 nm (n = 40). Furthermore, the S.D. map of the connexon surface (Fig. 2I, bottom) increased its central maxima now indicating that the channel entrance has further increased its flexibility. At the same time, the outer regions of the connexins were observed to slightly enhance flexibility, as indicated by their S.D. increasing to 0.2 nm (n = 40). Increasing the pH to 7.6 (Fig. 2D) finally widened the channel entrance (Fig. 2J, top) to a diameter of 1.7 ± 0.3 nm (n = 81). Concomitant with the opening of the channel entrance the S.D. map (Fig. 2J, bottom) showed that this structural region further increased its flexibility to a maximum S.D. of 0.35 nm (n = 40). Interestingly, individual connexins showed an increased S.D. of 0.25 nm at their outer rims as well. Further increasing the pH to 8 (Fig. 2E) and 8.5 (Fig. 2F) did not significantly change the correlation averages or connexon structures of the extracellular surface (data not shown). It should be noted that “partially closed channels” had a diameter lying between that observed for fully open and closed channels. AFM topographs showed that more than 85% of the single hemichannels had no significant deviation in their channel diameter (Fig. 3). This suggests that the partially closed hemichannels adopted a functional state that reflects an intermediate channel size between fully open and closed conformations and not a mixture of solely open and closed states.FIGURE 3Histogram of pH-induced changes of channel diameters taken from AFM topographs of single Cx26. Topographs were taken from Cx26 membranes in 10 mm HEPES-buffered solutions such as described in the legend to Fig. 2. At minimum, 80 connexins were measured for each histogram. The histogram distributions indicate that the pH-induced increase of the channel diameter is best represented by a process gradually switching from the closed to the fully open state.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To prove whether the observed conformational change was reversible, we decreased the pH to 6.0 after the channels were fully opened at pH 9.0. The AFM topographs recorded showed that the previously opened pore now re-closed, suggesting that their conformational change was fully reversible. Cycles of pH changes were often repeated more than four times. Table 1 summarizes our data for the measurements of inner pore diameter and channel entrance depth for different pH values.TABLE 1Summary of the data on the pH-dependent channel entrance maximum depth and channel entrance diameter measured at the extracellular connexon surfacepHChannel entrance maximum depthChannel entrance diameter (FWHM)nmnm5.5Not determined0.6 ± 0.36.00.4 ± 0.10.6 ± 0.36.50.5 ± 0.30.9 ± 0.37.01.0 ± 0.41.3 ± 0.27.61.1 ± 0.31.7 ± 0.38.00.9 ± 0.21.7 ± 0.38.50.9 ± 0.21.6 ± 0.3 Open table in a new tab Conformational Changes Are Not Dependent on HEPES Concentration—HEPES belongs to a class of aminosulfonate compounds that have been shown to act as modulators of the Cx26 channel (17Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Following the lead of Bevans and Harris (17Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we tested whether the observed conformational change is due solely to pH or to the binding of aminosulfonates to Cx26, which in turn modulates a pH-induced conformational change. We increased the HEPES concentration to 50 mm and imaged the extracellular connexon surface (Fig. 4). Surprisingly, AFM topographs recorded at pH 8.0 (Fig. 4A) and at pH 9 (Fig. 4B) showed that the open state of the connexon channels was not influenced by the increase in protonated HEPES. For both pH conditions, the averages of the inner channel diameters measured from single connexons were 1.6 ± 0.3 nm (n = 83), such as observed for the open connexon conformation (Figs. 2 and 3). Additional experiments were performed in an effort to close open channels with higher HEPES concentrations at pH ≥ 7.5. The sample was absorbed to the mica surface at 50 mm HEPES, pH 7.5, and AFM imaging was performed with the same buffer. At this pH, the effective concentration of protonated HEPES would be maximal at ∼25 mm. Under these conditions, the channels remained open. Open channels were also observed at HEPES concentrations up to 200 mm at pH 7.5 (data not shown). In each case, a positive control (10 mm HEPES, lowering the pH) was included to ensure the functionality of the sample. Alternatively, adsorption at pH 6.5 (50 mm HEPES) was performed to ensure HEPES binding and then increased the pH to 7.5 for AFM imaging. In this case, the topographs revealed open channels at pH 7.5 but partially closed channels at pH 6.5. Therefore, 10 mm may be the concentration for which binding is at saturation conditions in these experiments. No Conformational Change Occurs in the Absence of Aminosulfonates—According to previous findings (17Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3711-3719Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18Tao L. Harris A.L. J. Biol. Chem. 2004; 279: 38544-38554Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), the pH-dependent gating of Cx26 could be only observed in the presence of aminosulfonate-containing compounds. To test this hypothesis and to prove that the conformational change observed can be indeed correlated to the gating mechanism, we imaged connexin preparations at different pH, buffered in non-aminosulfonate buffers. AFM topographs of maleate-buffered connexons showed that they did not change their conformation at pH 6.0, 6.5, or 7.0 (Fig. 5, A–C). The connexon surface did not change at pH values higher than 7.0. Similarly, the extracellular connexon surface apparently did not show a pH-dependent change in their channel diameter if the aqueous solution was buffered with potassium phosphate (KH2PO4) (Fig. 5, D–F). All topographs (Fig. 5, A–F) and correlation averages (Fig. 5, G–N) have in common that the channel entrance appeared widely opened throughout the pH ranges imaged (compare with Fig. 2). Quantitative Analysis of the Channel Closure—The diameter of the average connexon decreased gradually with lower pH (Fig. 6A). Whereas the depth of the channel decreased concomitant with an increase in the width of the connexon lobes, the overall diameter of the connexon itself does not change (Fig. 6B). This is also reflected in the difference image between the fully closed (pH 6.0, Fig. 7A) and fully open (pH 7.6, Fig. 7B) averages shown in Fig. 7C. In this difference image, positive differences are displayed as red and negative differences as black. The medium red level reflects no differences. It is also clear from this difference image that the extracellular region of the subunits rotate by ∼6.5° between the open and closed states (Fig. 7, A and B) thereby changing the diameter of the pore. Our previously published hemichannel structure shows a narrowing of the connexon pore at the extracellular end that may be part of a physical gate closing upon acidification (36Perkins G.A. Goodenough D.A. Sosinsky G.E. Biophys. J. 1997; 72: 533-544Abstract Full Text PDF PubMed Scopus (77) Google Scholar).FIGURE 7Model of pH-induced cl" @default.
• W2049654315 created "2016-06-24" @default.
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• W2049654315 creator A5026602737 @default.
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• W2049654315 date "2007-03-01" @default.
• W2049654315 modified "2023-10-15" @default.
• W2049654315 title "Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels" @default.
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