HEK293S GnTI− cells were transiently transfected at a density of 2.0 × 106 cells ml−1, with the plasmids and FectoPRO (Polyplus). Approximately 360 μg of the CeCLHM-1 plasmid was premixed with 360 μl of FectoPRO reagent in 80 ml of fresh FreeStyle 293 medium for 10 to 20 min before transfection. For transfection, 80 ml of the mixture was added to 0.8 liters of cell culture and incubated at 37°C in the presence of 8% CO2. After 18 to 20 hours, 2.2 mM valproic acid was added, and the cells were further incubated at 30°C in the presence of 8% CO2 for 48 hours. The cells were collected by centrifugation (3000g, 10 min, 4°C) and disrupted by Dounce homogenization in hypotonic buffer [50 mM Hepes-NaOH (pH 7.5), 10 mM KCl, deoxyribonuclease (DNase) I (0.04 mg ml−1), and protease inhibitor cocktail].

Calcium homeostasis modulator (CALHM) family proteins are Ca²⁺-regulated adenosine triphosphate (ATP)–release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo–electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and Caenorhabditis elegans CLHM-1 at resolutions of 2.66, 3.4, and 3.6 Å, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD–intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.