Localization of the voltage-dependent anion channel-1 Ca2+-binding sites

Adrian Israelson, Salah Abu-Hamad, Hilal Zaid, Edna Nahon, Varda Shoshan-Barmatz

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca2+-binding proteins and to strongly inhibit their Ca2+-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca2+-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca2+, in the presence of 1 M NaCl, but not Mg2+, prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [103Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca2+-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca2+-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca2+-binding proteins and localizing their Ca2+-binding sites.

Original languageEnglish
Pages (from-to)235-244
Number of pages10
JournalCell Calcium
Volume41
Issue number3
DOIs
StatePublished - 1 Jan 2007

Keywords

  • Apoptosis
  • Azido ruthenium (AzRu)
  • Ca-binding sites
  • Porin
  • Ruthenium red (RuR)
  • Voltage-dependent anion channel (VDAC)

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