Localization of the voltage-dependent anion channel-1 Ca2+-binding sites.
Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca(2+)-binding proteins and to strongly inhibit their Ca(2+)-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca(2+)-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca(2+), in the presence of 1M NaCl, but not Mg(2+), prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [(103)Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca(2+)-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 Ca(2+)-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 Ca(2+)-binding proteins and localizing their Ca(2+)-binding sites.
Israelson
Adrian
A
Department of Life Sciences and The Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Abu-Hamad
Salah
S
Zaid
Hilal
H
Nahon
Edna
E
Shoshan-Barmatz
Varda
V
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D009942:Organometallic Compounds / Q000494:pharmacology; D010455:Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014466:Ultraviolet Rays; D050995:Voltage-Dependent Anion Channel 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D009942:Organometallic Compounds; D010455:Peptides; C494763:Vdac1 protein, mouse; C508266:azido ruthenium; D050995:Voltage-Dependent Anion Channel 1; D002118:Calcium