The Voltage Gates of Connexin Channels are Sensitive to CO2

Peracchia, Camillo; Young, Kate; Wang, Xiao; Chen, Joey; Peracchia, Lillian

doi:10.1080/714040433

Cited by 3 publications

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“…Typically, CO 2 has been used as the acidification agent in whole cell experiments, and the term is often used interchangeably with pH gating (6). It has been proposed that calmodulin plays a role in modulating chemical gating (44) by acting as a "cork" to plug the channel entrance at the cytoplasmic surface. Here, we imaged the effects of direct acidification in the presence of different buffers rather than with CO 2 .…”

Section: Volume 282 • Number 12 • March 23 2007mentioning

confidence: 99%

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Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Bippes

Hand

et al. 2007

Journal of Biological Chemistry

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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...

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Section: Volume 282 • Number 12 • March 23 2007mentioning

confidence: 99%

“…pH Gating of Connexins-Chemical gating has been extensively studied in cell-cell channels (43,44). Typically, CO 2 has been used as the acidification agent in whole cell experiments, and the term is often used interchangeably with pH gating (6).…”

Section: Volume 282 • Number 12 • March 23 2007mentioning

confidence: 99%

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Bippes

Hand

et al. 2007

Journal of Biological Chemistry

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“…The open probability (P o ) of hemichannels is modulated by intracellular and extracellular signals, including plasma membrane voltage (5), extracellular divalent ion concentration (32,35), cytoplasmic pH (15,17), phosphorylation (1,2) and redox potential (19 -22). Regulation of ion channel gating involving direct interactions between neighboring ion channels (channel cooperativity) has been described (7)(8)(9).…”

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confidence: 99%

Voltage-dependent facilitation of Cx46 hemichannels

Retamal

Yin

Altenberg

et al. 2010

American Journal of Physiology-Cell Physiology

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Gap junction channels are formed by two hemichannels in series (one from each neighboring cell), which are in turn connexin hexamers. Under normal conditions, hemichannels at the plasma membrane are mostly closed but can be opened by changes in membrane voltage, extracellular divalent ion concentration, phosphorylation, pH, and redox potential. Recently, interactions between channels have been found to modulate the activity of several ion channels, including gap junction channels. Here, we studied whether connexin46 (Cx46) hemichannels display such behavior. We studied the response of the Cx46 hemichannels expressed in Xenopus laevis oocytes to consecutive depolarization pulses. Hemichannels formed by wild-type Cx46 and a COOH-terminal domain truncation mutant (Cx46DeltaCT) were activated by voltage pulses. When the hemichannels were depolarized repeatedly from -60 mV to +80 mV, the amplitude of the outward and tail currents increased progressively with successive pulses. This phenomenon ("current facilitation") depended on the amplitude of the depolarization, reaching a maximum at approximately +60 mV in oocytes expressing Cx46, and on the interval between pulses, disappearing with intervals longer than about 20 s. The current facilitation was also present in oocytes expressing Cx46DeltaCT, ruling out a primary role of this domain in the facilitation. Nominal removal of divalent cations from the extracellular side caused maximal current activation of Cx46 and Cx46DeltaCT hemichannels and prevented facilitation. The results suggest that Cx46 hemichannels show a cooperative activation independent of their COOH-terminal domain.

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Inversion of both gating polarity and CO₂ sensitivity of voltage gating with D3N mutation of Cx50

Peracchia¹,

Peracchia²

2005

American Journal of Physiology-Cell Physiology

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The effect of CO 2-induced acidification on transjunctional voltage (Vj) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO2 application greatly decreased the Vj sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO2 also affected the kinetics of Vj dependent inactivation of junctional current (Ij), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO2 sensitivity of chemical gating such that even CO2 concentrations as low as 2.5% significantly lowered junctional conductance (Gj). With Cx50 channels Gj dropped by 78% with a drop in intracellular pH (pHi) to 6.83, whereas with Cx50-D3N channels Gj dropped by 95% with a drop in pHi to just 7.19. We have previously hypothesized that the way in which Vj gating reacts to CO2 might be related to connexin's gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO2 on Vj gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes GAP JUNCTIONS are regions of cell contact that mediate the exchange of small cytosolic molecules via cell-cell channels. A gap junction channel results from the extracellular interaction of two hemichannels (connexons), each a hexamer of connexin proteins. Connexins (Cx) contain four transmembrane domains, two extracellular loops, a cytoplasmic loop, a short NH 2 terminus (NT), and a COOH terminus of variable length. The sequences of cytoplasmic loop and COOH terminus vary significantly among the members of the connexin family, whereas those of the other domains are relatively well conserved (see Ref. 14 for review).Gap junction channels are gated by transjunctional voltage (V j ; 27) and increased intracellular [Ca 2ϩ ] (10, 25) or [H ϩ ] i (28, 33) via molecular mechanisms that are still poorly defined (see Ref. 12 for review). At least two V j -sensitive gates have been identified: fast and slow. On the basis of their behavior at the single channel level, fast V j gate and chemical gate are believed to be distinct: the former closes rapidly (Ͻ1 ms) but incompletely, leaving a 20 -30% residual conductance, whereas the latter closes slowly (8 -10 ms) but completely (5). In contrast, slow V j gate and chemical gate are likely to be the same (5,17,20). Slow and fast V j gates are in series, and each hemichannel appears to have both gates. The slow gate closes at the negative side of V j in all connexin channels tested, whereas the polarity of the fast V j gating mechanism varies among connexin channels (see Ref. 8 for review).In the past, chemical and V j gating have been studied almost exclusively by testing chemicals and V j gradients, respectively, whereas minimal attention has been ...

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The Voltage Gates of Connexin Channels are Sensitive to CO2

Cited by 3 publications

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Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Voltage-dependent facilitation of Cx46 hemichannels

Inversion of both gating polarity and CO₂ sensitivity of voltage gating with D3N mutation of Cx50

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The Voltage Gates of Connexin Channels are Sensitive to CO2

Cited by 3 publications

References 0 publications

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

Voltage-dependent facilitation of Cx46 hemichannels

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

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Inversion of both gating polarity and CO₂ sensitivity of voltage gating with D3N mutation of Cx50