Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome

Helmuth A. Sanchez, Rick Bienkowski, Nefeli Slavi, Miduturu Srinivas, Vytautas Verselis

Research output: Contribution to journalArticle

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Abstract

Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca 2+. This study presents two new possible contributing factors, pH and Zn2+. Plasma pH levels and micromolar concentrations of Zn 2+ inhibit WTCx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H+ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation bypH or Zn2+. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn2+ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn2+. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to closeCx26 hemichannels via this gating mechanism.

Original languageEnglish (US)
Pages (from-to)21519-21532
Number of pages14
JournalJournal of Biological Chemistry
Volume289
Issue number31
DOIs
StatePublished - Aug 1 2014

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Acidification
Mutation
Protonation
Cell membranes
Sulfhydryl Compounds
Buffers
Crystal structure
Modulation
Fluxes
Membranes
Plasmas
Sulfhydryl Reagents
Keratitis-Ichthyosis-Deafness Syndrome
Cell Membrane
succinimide

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

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Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome. / Sanchez, Helmuth A.; Bienkowski, Rick; Slavi, Nefeli; Srinivas, Miduturu; Verselis, Vytautas.

In: Journal of Biological Chemistry, Vol. 289, No. 31, 01.08.2014, p. 21519-21532.

Research output: Contribution to journalArticle

Sanchez, Helmuth A. ; Bienkowski, Rick ; Slavi, Nefeli ; Srinivas, Miduturu ; Verselis, Vytautas. / Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 31. pp. 21519-21532.
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abstract = "Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca 2+. This study presents two new possible contributing factors, pH and Zn2+. Plasma pH levels and micromolar concentrations of Zn 2+ inhibit WTCx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H+ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation bypH or Zn2+. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn2+ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn2+. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to closeCx26 hemichannels via this gating mechanism.",
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