Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

Abstract

Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x L ) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x L (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x L proapoptotic. Transfection of double knockout (Bax À/À /Bak À/À ) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x L E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.

Dimers of mitochondrial ATP synthase form the permeability transition pore

“…Planar lipid bilayer experiments were performed as described (51). Briefly, bilayers of about 150-200 pF capacity were prepared using purified soybean azolectin.…”

F-ATPase of Drosophila melanogaster Forms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+ Release

“…18. Briefly, bilayers of 150 -200 picofarads capacitance were prepared using purified soybean asolectin.…”

Targeting the Potassium Channel Kv1.3 Kills Glioblastoma Cells

“…Mechanistically, a functional interaction between Bax and the channel protein was found [9], which was determined by a positively charged conserved lysine at position 128 of Bax protruding into the intermembrane space facing the IMM after Bax insertion into the outer mitochondrial membrane [10]. Mutation of this amino acid abrogated the apoptotic effects of Bax in both isolated mitochondria and in intact cells expressing the mutated protein [9,11]. Since then, specific membrane permeant inhibitors, including the Ruta graveolens plant derived psoralens Psora-4, and PAP-1, the anti-mycobacterial drug clofazimine and novel mitochondrial targeted PAP-1 derivatives, namely PAPTP and PCARBTP, synthesized by our group, were used to mimic the interactions of Bax and mitoKv1.3 in different cancer cell lines that express Kv1.3 [12,13].…”