A cell relies on proteins, termed ion channels, that facilitate the passage of ions across its membranes. These channels contain pores that activate in response to certain stimuli, allowing ion flow in their open state. Previous research has predominantly focused on ion channels with narrow and selective pores, which permit only certain types of inorganic ions to cross the membrane. However, there is increasing interest in proteins that form channels with wider pores that facilitate the permeation of larger substrates. These large-pore channels play various biological roles by allowing signaling molecules such as ATP to cross the membrane. One family of large-pore channels is the recently discovered calcium homeostasis modulators (CALHM) family, which contains six paralogs in humans. CALHM1 has been shown to form ion channels that are activated by voltage and the decrease of extracellular calcium. It pairs with CALHM3 to form heteromeric channels with improved activation properties. CALHM1 and 3 have been found in primate taste buds, and their role in perceiving sweet, bitter, and umami tastes through ATP efflux and subsequent purinergic signaling has been demonstrated. Despite these breakthroughs, the mechanisms underlying ion permeation and gating of CALHM channels, and the function of other family members have remained elusive. In this thesis project, I aimed to study the functional and structural features of human CALHM proteins, with a particular focus on uncharacterized members of the family. To achieve this goal, I have identified biochemically stable constructs of CALHM2, CALHM4, and CALHM6 and used cryo-electron microscopy to characterize their detailed three-dimensional structures. These findings provided the first structures of CALHM4 and CALHM6, revealing their unique features to assemble as large channels composed of ten or eleven subunits with a central pore. Strikingly, I observed two different conformations between the homologues that significantly impacted pore size and geometry, potentially suggesting a gating mechanism. In one conformation, the pore-lining α-helix 1 runs parallel to the pore axis, creating a wide-open cylindrical pore, while in the other conformation, the same elements lifts to form a conical pore, which narrows towards the cytoplasm. Additionally, the CALHM4 structure exhibited extra bilayer-like density inside the pore, which likely either originates from detergents used in the purification or co-purified lipids, indicating a potential role of lipids in the regulation of channel activity. Although the physiological relevance of observed features remains unclear, the described work has provided valuable insight into the general architecture and potential activation mechanisms of the CALHM channels. Complementary functional studies of CALHM2, CALHM4, and CALHM6 by electrophysiology and in vitro transport assays did not show any activity under conditions that activate CALHM1, suggesting a distinct mechanism of activation. Transcript analysis demonstrated that all three homologues are highly enriched in the placenta, prompting the investigation of mutual interactions in vitro. I could show that CALHM2 and CALHM4 interact to form heteromeric CALHM channels, similar to the previously reported CALHM1/3 heteromers. However, functional studies did not show any activity in cells co-expressing CALHM2 and CALHM4, in contrast to the altered phenotype of 2 CALHM1/3 heteromers and similar to the lack of activity that was previously observed in homomeric CALHM2, 4 and 6 channels. Despite the observed lack of activity, I pursued the structural characterization of heteromeric CALHM2/4 complexes in the hope to uncover structural features that facilitate the comprehension of CALHM function. To overcome the challenge of identifying structurally similar subunits in a heteromeric channel, I have generated specific sybodies to each of the two homologues to facilitate their distinction during reconstruction. Before structure determination of heteromeric channel complexes, I first characterized the homomeric CALHM paralogs in complex with their respective sybodies, which identified their respective binding sites. These efforts also yielded a structure of CALHM2, which was prevented in case of the apo-protein due to the preferred channel orientation on cryo-EM grids. CALHM2, like CALHM4 and 6, form large channels and its pore conformation is similar as observed for CALHM6. The structure of CALHM2/4 complexes revealed heteromeric channels with broad distribution of oligomeric states, ranging from decamers to dodecamers, with varying subunit stoichiometries. The high-resolution structure of a predominant population showed a heteromeric channel with an excess of CALHM2 subunits that are arranged in a cluster and a group of two to three CALHM4 subunits. While most subunits reside in the preferred conformation defined in homomeric structures, the conformation of a CALHM2 subunit, located in the neighborhood of CALHM4, differed in conformation, aiding the understanding on the conversion of states. Intriguingly, similar to the CALHM4 structure, extra density was observed in a section of the pore close to the subunits residing in cylindrical conformations. This further supports the notion that the pore-lining α-helix and lipids may play a crucial role in the gating of CALHM channels, which likely employ complex regulatory mechanisms that may be distinct for different members of the family
