Rapid and direct effects of pH on connexins revealed by the connexin 46 hemichannel preparation

E. Brady Trexler, Feliksas F. Bukauskas, Michael V. L. Bennett, Thaddeus A. Bargiello, Vytautas Verselis

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting (~10-7 M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.

Original languageEnglish (US)
Pages (from-to)721-742
Number of pages22
JournalJournal of General Physiology
Volume113
Issue number5
DOIs
StatePublished - May 1999

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Connexins
connexin 46
Gap Junctions

Keywords

  • Connexin
  • Gating
  • Intercellular communication
  • Ion channel
  • pH

ASJC Scopus subject areas

  • Physiology

Cite this

Rapid and direct effects of pH on connexins revealed by the connexin 46 hemichannel preparation. / Trexler, E. Brady; Bukauskas, Feliksas F.; Bennett, Michael V. L.; Bargiello, Thaddeus A.; Verselis, Vytautas.

In: Journal of General Physiology, Vol. 113, No. 5, 05.1999, p. 721-742.

Research output: Contribution to journalArticle

Trexler, E. Brady ; Bukauskas, Feliksas F. ; Bennett, Michael V. L. ; Bargiello, Thaddeus A. ; Verselis, Vytautas. / Rapid and direct effects of pH on connexins revealed by the connexin 46 hemichannel preparation. In: Journal of General Physiology. 1999 ; Vol. 113, No. 5. pp. 721-742.
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AB - pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting (~10-7 M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.

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