title: Identification of Bax-voltage-dependent anion channel 1 complexes in digitonin-solubilized cerebellar granule neurons.
abstract: Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane-associated Bax has primarily been studied using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage-dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co-immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two-dimensional blue native-SDS-PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co-precipitated VDAC1, indicating the co-precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax-VDAC1 interactions that may be disrupted with CHAPS.
authors.affiliation: Department of Biology, University of Mississippi, University, Mississippi, USA.
authors.forename: Dennis B
authors.initials: DB
authors.lastname: Huckabee
authors.forename: Mika B
authors.initials: MB
authors.lastname: Jekabsons
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002525:Cerebellar Cortex / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002793:Cholic Acids / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D009474:Neurons / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D061251:Primary Cell Culture; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000502:physiology
chemical_list: C494828:Bax protein, rat; D002793:Cholic Acids; C494764:Vdac1 protein, rat; D051028:bcl-2-Associated X Protein; D050995:Voltage-Dependent Anion Channel 1; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate