Bifunctional adenylate cyclase-haemolysin toxin precursor, CyaA.  Although homologous, HlyA (1.C.11.1.3) and CyaA  exhibit different modes of permeabilization (Fiser and Konopásek 2009).  A pore model comprising three alpha2-loop-alpha3 hairpins suggested that Gly530XXGly533XXXGly537  in TMS2 could function in toxin oligomerization (Juntapremjit et al. 2015).  Structural integrity of TMSs 1, 2, 3 and 5, but not 4, is important for haemolytic activity, particularly for transmembrane helices 2 and 3 that might form the pore (Powthongchin and Angsuthanasombat 2009). CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis (Wald et al. 2016).  The toxin penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into the cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, the ~1300 residue-long RTX hemolysin moiety of CyaA permeabilizes target cell membranes for efflux of cytosolic potassium ions (Svedova et al. 2016).  Positively-charged side-chains substituted at positions Gln574 and Glu581 in the pore-lining alpha3 enhance hemolytic activity and ion-channel opening, mimicing the highly-active RTX (repeat-in-toxin) cytolysins (Kurehong et al. 2017). Residues 529 to 549 participate in membrane penetration and pore-forming activity (Roderova et al. 2019). Two distinct conformers of CyaA appear to accomplish its two parallel activities within target cell membranes. The translocating conformer would deliver the N-terminal adenylyl cyclase domain into the cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Two clusters of negatively charged residues within this linking segment (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA as a function of the calcium concentration (Sukova et al. 2020). Four cholesterol-recognition motifs in the pore-forming and translocation domains of CyaA are essential for invasion of eukaryotic cells and lysis of erythrocytes (Amuategi et al. 2022).